Oxa-and thiadiazoles and their use as metalloproteinase inhibitors

ABSTRACT

Compounds formula (IA) or (IB), wherein W represents HO(C═O)—, HONH(C═O)— or H(C═O)N(OH)—; X represents —O— or —S—; and R 1 , R 2 , and R 3  are as defined in the description and claims, are inhibitors of matrix metal oproteinases, in particular MMP9 and/or MMP12.

The present invention relates to therapeutically active hydroxamic andcarboxylic acid derivatives, to processes for their preparation, topharmaceutical compositions containing them, and to the use of suchcompounds in medicine. In particular, the compounds are inhibitors ofmatrix metalloproteinases.

BACKGROUND TO THE INVENTION

The matrix metalloproteinases (MMP's) are a family of zinc containingendopeptidases which are capable of cleaving large biomolecules such asthe collagens, proteoglycans and gelatins. Imbalance between active MMPsand endogenous inhibitors, leads to excessive tissue disruption. Thethree main groups of MMPs are the collagenases, the gelatinases, and thestromelysins. Collagenases include fibroblast collagenase (MMP-1),neutrophil collagenase (MMP-8), and collagenase 3 (MMP-13). Gelatinasesinclude 72 kDa gelatinase (gelatinase A; MMP-2) and 92 kDa gelatinase(gelatinase B; MMP-9). Stromelysins include stromelysin 1 (MMP-3),stromelysin 2 (MMP-10) and matrilysin (MMP-7). However there are MMPswhich do not fit neatly into the above groups, for examplemetalloelastase (MMP-12), membrane-type MMP (MT-MMP or MMP-14) andstromelysin 3 (MMP-11).

Over-expression and activation of MMPs have been linked with a widerange of diseases such as cancer; rheumatoid arthritis; osteoarthritis;chronic inflammatory disorders, such as asthma, bronchitis andemphysema; cardiovascular disorders, such as atherosclerosis; cornealulceration; dental diseases such as gingivitis and periodontal disease;neurological disorders, such as multiple sclerosis and restenosis. Forexample, MMP-12 is required for the development of cigarettesmoke-induced emphysema in mice, Science, 277, 2002 (1997). Inhibitionof MMPs is therefore a strategy for treatment of such disease states.However, there is evidence that non-selective inhibition of matrixmetalloproteinase activity may affect normal physiological processleading to dose limiting side effects. Selective inhibition of MMP-12and/or MMP-9 is thought to be a particularly relevant strategy forintervention in inflammatory conditions.

MMPs can hydrolyse the membrane-bound precursor of the pro-inflammatorycytokine tumour necrosis factor a (TNF-a). This cleavage yields maturesoluble TNF-a and the inhibitors of MMPs can block production of TNF-aboth in vitro and in vivo. This pharmacological action is a probablecontributor to the anti-inflammatory action of this class of compounds.

For a recent review of MMP inhibition as reflected in the patentliterature, see Doherty et. Al. Therapeutic Developments in MatrixMetalloproteinase Inhibition; Expert Opinions on Therapeutic Patents,2002, 12, 665-707.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides a class of compounds which are inhibitorsof MMPs. The class includes compounds which are selective inhibitors ofMMP12 relative to the collagenases and stromelysins. In addition,compounds of the invention can exhibit selective activity towards MMP-9.Compounds of the invention are therefore indicated for treatment ofdiseases primarily mediated by MMP-9 and/or MMP-12, especiallyinflammatory conditions such as multiple sclerosis and fibrosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph illustrating the average ASAT and ALAT levels forcontrol animals and those treated with the compound of Example 13 atthree different dosages.

FIG. 2 is a bar graph illustrating the average area percentages offibrosis in the livers of animals.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention there is provided compound formula(IA or (IB))

wherein

-   w represents HO(C═O)—, HONH(C═O)— or H(C═O)N(OH)—;-   X represents —O— or —S—;-   R₁ represents    -   hydrogen;    -   —OH or —SH;    -   fluoro or chloro;    -   —CF₃;    -   (C₁-C₆)alkyl;    -   (C₁-C₆)alkoxy;    -   (C₂-C₆)alkenyl;    -   phenyl or substituted phenyl;    -   phenyl (C₁-C₆)alkyl or substituted phenyl(C₁-C₆)alkyl;    -   phenyl (C₂-C₆)alkenyl or substituted phenyl(C₂-C₆)alkenyl        heterocyclyl or substituted heterocyclyl;    -   heterocyclyl(C₁-C₆)alkyl or substituted heterocyclyl        (C₁-C₆)alkyl;    -   a group BSO_(n)A- wherein n is 0, 1 or 2 and B is hydrogen or a        (C₁-C₆) alkyl, phenyl, substituted phenyl, heterocyclyl        substituted heterocyclyl, (C₁-C₆)acyl, phenacyl or substituted        phenacyl group, and A represents (C₁-C₆)alkylene;    -   —NH₂, (C₁-C₆)alkylamino or di(C₁-C₆)alkylamino;    -   amino(C₁-C₆)alkyl, (C₁-C₆)alkylamino(C₁-C₆)alkyl,        di(C₁-C₅)alkylamino(C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl,        mercapto(C₁-C₆)alkyl or carboxy(C₁-C₆) alkyl wherein the amino-,        hydroxy-, mercapto- or carboxyl-group are optionally protected        or the carboxyl-group amidated; or    -   a cycloalkyl, cycloalkenyl or non-aromatic heterocyclic ring        containing up to 3 heteroatoms, any of which may be (i)        substituted by one or more substituents selected from C₁-C₆        alkyl, C₂-C₆ alkenyl, halo, cyano (—CN), —CO₂H, —CO₂R, —CONH₂,        —CONHR, —CON(R)₂, —OH, —OR, oxo-, —SH, —SR, —NHCOR, and —NHCO₂R        wherein R is C₁-C₆ alkyl or benzyl and/or (ii) fused to a        cycloalkyl or heterocyclic ring;-   R₂ represents a group R₁₀—(X)_(n)-(ALK)_(m)— wherein    -   R₁₀ represents hydrogen, or a C₁-C₅ alkyl, C₂-C₆ alkenyl, C₂-C₆        alkynyl, cycloalkyl, aryl, or heterocyclyl group, any of which        may be unsubstituted or substituted by (C₁-C₁₂)alkyl,        (C₁-C₁₂)alkoxy, hydroxy, mercapto, (C₁-C₁₂)alkylthio, amino,        halo (including fluoro, chloro, bromo and iodo),        trifluoromethyl, cyano, nitro, oxo, —COOH, —CONH₂, —COOR^(A),        —NHCOR^(A), —CONHR^(A), —NHR^(A), —NR^(A)R^(B), or CONR^(A)R^(B)        wherein R^(A) and R^(B) are independently a (C₁-C₆)alkyl group        and    -   ALK represents a straight or branched divalent C₁-C₆ alkylene,        C₂-C₆ alkenylene, or C₂-C₅ alkynylene radical, and may be        interrupted by one or more non-adjacent —NH—, —O— or        —S-linkages,    -   X represents —NH—, —O—, —S—, —NR^(C) or —NCOR^(C) wherein R^(C)        is a (C₁-C₁₂)alkyl group and    -   m and n are independently 0 or 1;-   R₃ represents the side chain of a natural or non-natural alpha amino    acid;-   R₄ represents optionally substituted    -   C₁-C₆ alkyl,    -   C₂-C₆ alkenyl,    -   C₂-C₆ alkynyl, C₁-C₃ perfluoroalkyl,    -   cycloalkyl,    -   cycloalkyl(C₁-C₆ alkyl)-,    -   cycloalkenyl,    -   cycloalkenyl(C₁-C₆ alkyl)-,    -   phenyl,    -   phenyl(C₁-C₆ alkyl)-,    -   naphthyl,    -   non-aryl heterocyclyl,    -   non-aryl heterocyclyl(C₁-C₆ alkyl)-,    -   heteroaryl; or    -   heteroaryl(C₁-C₆ alkyl)-;        and pharmaceutically acceptable salts hydrates and solvates        thereof.

As used herein the term “(C₁-C₆)alkyl” means a straight or branchedchain alkyl moiety having from 1 to 6 carbon atoms, including forexample, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, t-butyl, n-pentyl and n-hexyl.

As used herein the term “divalent (C₁-C₆)alkylene radical” means asaturated hydrocarbon chain having from 1 to 6 carbon atoms and twounsatisfied valences.

As used herein the term “(C₂-C₆)alkenyl” means a straight or branchedchain alkenyl moiety having from 2 to 6 carbon atoms having at least onedouble bond of either E or Z stereochemistry where applicable. The termincludes, for example, vinyl, allyl, 1- and 2-butenyl and2-methyl-2-propenyl.

As used herein the term “divalent (C₂-C₆)alkenylene radical” means ahydrocarbon chain having from 2 to 6 carbon atoms, at least one doublebond, and two unsatisfied valences.

As used herein the term “C₂-C₆ alkynyl” refers to straight chain orbranched chain hydrocarbon groups having from two to six carbon atomsand having in addition one triple bond. This term would include forexample, ethynyl, 1-propynyl, 1- and 2-butynyl, 2-methyl-2-propynyl,2-pentynyl, 3-pentynyl, 4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl and5-hexynyl.

As used herein the term “divalent (C₂-C₆)alkynylene radical” means ahydrocarbon chain having from 2 to 6 carbon atoms, at least one triplebond, and two unsatisfied valences.

As used herein the term “cycloalkyl” means a saturated alicyclic moietyhaving from 3-8 carbon atoms and includes, for example, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

As used herein the term “cycloalkenyl” means an unsaturated alicyclicmoiety having from 3-8 carbon atoms and includes, for example,cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyland cyclooctenyl. In the case of cycloalkenyl rings of from 5-8 carbonatoms, the ring may contain more than one double bond.

As used herein the term “aryl” refers to a mono-, bi- or tri-cycliccarbocyclic aromatic group, and to groups consisting of two covalentlylinked monocyclic carbocyclic aromatic groups. Illustrative of suchgroups are phenyl, biphenyl and napthyl.

As used herein the unqualified term “heterocyclyl” or “heterocyclic”includes “heteroaryl” as defined below, and in particular means a 5-8membered aromatic or non-aromatic heterocyclic ring containing one ormore heteroatoms selected from S, N and O, and optionally fused to abenzyl or second heterocyclic ring, and the term includes, for example,pyrrolyl, furyl, thienyl, piperidinyl, imidazolyl, oxazolyl, thiazolyl,thiadiazolyl, thiazepinyl, pyrazolyl, pyridinyl, pyrrolidinyl,pyrimidinyl, morpholinyl, piperazinyl, indolyl, and benzimidazolylrings.

As used herein the term “heteroaryl” refers to a 5- or 6-memberedaromatic ring containing one or more heteroatoms, and optionally fusedto a benzyl or pyridyl ring; and to groups consisting of two covalentlylinked 5- or 6-membered aromatic rings each containing one or moreheteroatoms; and to groups consisting of a monocyclic carbocyclicaromatic group covalently linked to a 5- or 6-membered aromatic ringscontaining one or more heteroatoms. Illustrative of such groups arethienyl, furyl, pyrrolyl, imidazolyl, benzimidazolyl, thiazolyl,pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl,oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,4-([1,2,3]-thiadiazoly-4-yl)phenyl and 5-isoxazol-3-ylthienyl.

As used herein the unqualified term “carbocyclyl” or “carbocyclic”refers to a 5-8 membered ring whose ring atoms are all carbon.

Unless otherwise specified in the context in which it occurs, the term“substituted” as applied to any moiety herein means substituted with upto four substituents, each of which independently may be (C₁-C₆)alkyl,phenyl, benzyl, (C₁-C₆)alkoxy, phenoxy, hydroxy, mercapto,(C₁-C₆)alkylthio, amino, halo (including fluoro, chloro, bromo andiodo), trifluoromethyl, cyano, nitro, oxo, —COOH, —CONH₂, —COR^(A),—COOR^(A), —NHCOR^(A), —CONHR^(A), NHR^(A), —NR^(A)R^(B), or—CONR^(A)R^(B) wherein R^(A) and R^(B) are independently a (C₁-C₆)alkylgroup. In the case where “substituted” means substituted by benzyl, thephenyl ring thereof may itself be substituted with any of the foregoing,except phenyl or benzyl.

As used herein the terms “side chain of a natural alpha-amino acid” and“side chain of a non-natural alpha-amino acid” mean the group R^(x) inrespectively a natural and non-natural amino acid of formulaNH₂—CH(R^(x))—COOH.

Examples of side chains of natural alpha amino acids include those ofalanine, arginine, asparagine, aspartic acid, cysteine, cystine,glutamic acid, histidine, 5-hydroxylysine, 4-hydroxyproline, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine, α-aminoadipic acid, α-amino-n-butyricacid, 3,4-dihydroxyphenylalanine, homoserine, α-methylserine, ornithine,pipecolic acid, and thyroxine.

In natural alpha-amino acid side chains which contain functionalsubstituents, for example amino, carboxyl, hydroxy, mercapto, guanidyl,imidazolyl, or indolyl groups as in arginine, lysine, glutamic acid,aspartic acid, tryptophan, histidine, serine, threonine, tyrosine, andcysteine, such functional substituents may optionally be protected.

Likewise, in the side chains of non-natural alpha amino acids whichcontain functional substituents, for example amino, carboxyl, hydroxy,mercapto, guanidyl, imidazolyl, or indolyl groups, such functionalsubstituents may optionally be protected.

The term “protected” when used in relation to a functional substituentin a side chain of a natural or non-natural alpha-amino acid means aderivative of such a substituent which is substantially non-functional.The widely used handbook by T. W. Greene and P. G. Wuts “ProtectiveGroups in Organic Synthesis” Second Edition, Wiley, New York, 1991reviews the subject. For example, carboxyl groups may be esterified (forexample as a C₁-C₆ alkyl ester), amino groups may be converted to amides(for example as a NHCOC₁-C₆ alkyl amide) or carbamates (for example asan NHC(═O)OC₁-C₆ alkyl or NHC(═O)OCH₂Ph carbamate), hydroxyl groups maybe converted to ethers (for example an OC₁-C₆ alkyl or a O(C₁-C₆alkyl)phenyl ether) or esters (for example a OC(═O)C₁-C₆ alkyl ester)and thiol groups may be converted to thioethers (for example atert-butyl or benzyl thioether) or thioesters (for example a SC(═O)C₁-C₆alkyl thioester).

There are at least two actual or potential chiral centres in thecompounds according to the invention because of the presence ofasymmetric carbon atoms. The presence of several asymmetric carbon atomsgives rise to a number of diastereoisomers with R or S stereochemistryat each chiral centre. The invention includes all such diastereoisomersand mixtures thereof. Currently, the preferred stereo configuration ofthe carbon atom carrying the R₂ group is R; that of the carbon atomcarrying the R₁ group (when asymmetric) is R; and that of the carbonatom carrying the R₃ group (when asymmetric) is S.

The group R₁R₁ may be, for example,

-   -   hydrogen, hydroxy, methyl, methoxy, trifluoromethyl, ethyl,        n-propyl, allyl phenylpropyl, cyclopropylmethyl,        phenylprop-2-enyl, thienylsulphanylmethyl,        thienylsulphinylmethyl, or thienylsulphonylmethyl; or    -   C₁-C₄ alkyl, eg methyl, ethyl n-propyl or n-butyl, substituted        by a phthalimido, 1,2-dimethyl-3,5-dioxo-1,2,4-triazolidin-4-yl,        3-methyl-2,5-dioxo-1-imidazolidinyl,        3,4,4-trimethyl-2,5-dioxo-1-imidazolidinyl,        2-methyl-3,5-dioxo-1,2,4-oxadiazol-4-yl,        3-methyl-2,4,5-trioxo-1-imidazolidinyl,        2,5-dioxo-3-phenyl-1-imidazolidinyl, 2-oxo-1-pyrrolidinyl,        2,5-dioxo-1-pyrrolidinyl or 2,6-dioxopiperidinyl,        5,5-dimethyl-2,4-dioxo-3-oxazolidinyl,        hexahydro-1,3-dioxopyrazolo[1,2,a][1,2,4]-triazol-2-yl, or a        naphththalimido (i.e.        1,3-dihydro-1,3-dioxo-2H-benz[f]isoindol-2-yl),        1,3-dihydro-1-oxo-2H-benz[f]isoindol-2-yl,        1,3-dihydro-1,3-dioxo-2H-pyrrolo[3,4-b]quinolin-2-yl, or        2,3-dihydro-1,3-dioxo-1H-benz[d,e]isoquinolin-2-yl group; or    -   cyclohexyl, cyclooctyl, cycloheptyl, cyclopentyl, cyclobutyl,        cyclopropyl, tetrahydropyranyl or morpholinyl.

Presently preferred R₁ groups include hydrogen, hydroxy, methoxy,cyclopentyl, n-propyl, and allyl. Of these, hydrogen, hydroxy, methoxyand allyl are presently more preferred.

The group R₂R₂ may for example be

-   -   C₁-C₁₂ alkyl, C₃-C₆ alkenyl or C₃-C₆ alkynyl;    -   cycloalkyl(C₁-C₆ alkyl)-;    -   phenyl(C₁-C₆ alkyl)-, phenyl(C₃-C₆ alkenyl)- or phenyl(C₃-C₆        alkynyl)-optionally substituted in the phenyl ring;    -   heteroaryl(C₁-C₆ alkyl)-, heteroaryl(C₃-C₆ alkenyl)- or        heteroaryl(C₃-C₆ alkynyl)-optionally substituted in the        heteroaryl ring;    -   4-phenylphenyl(C₁-C₆ alkyl)-, 4-phenylphenyl(C₃-C₆ alkenyl)-,        4-phenylphenyl(C₃-C₆ alkynyl)-, 4-heteroarylphenyl(C₁-C₆        alkyl)-, 4-heteroarylphenyl(C₃-C₆ alkenyl)-,        4-heteroarylphenyl(C₃-C₆ alkynyl)-, optionally substituted in        the terminal phenyl or heteroaryl ring;    -   phenoxy(C₁-C₆ alkyl)- or heteroaryloxy(C₁-C₆ alkyl)-optionally        substituted in the phenyl or heteroaryl ring;

Specific examples of such groups include methyl, ethyl, n- oriso-propyl, n-, iso- or tert-butyl, n-pentyl, n-hexyl, n-heptyl,n-nonyl, n-decyl, prop-2-yn-1-yl, cyclohexylethyl, cyclopentylmethyl,3-phenylprop-2-yn-1-yl, 3-(2-chlorophenyl)prop-2-yn-1-yl, benzylphenylpropyl, 4-chlorophenylpropyl, 4-methyl phenylpropyl,4-methoxyphenylpropyl, phenoxybutyl, 3-(4-pyridylphenyl)propyl-,3-(4-(4-pyridyl)phenyl)prop-2-yn-1-yl, 3-(4-phenylphenyl)propyl-,3-(4-phenyl)phenyl)prop-2-yn-1-yl and 3-[(4-chlorophenyl)phenyl]propyl-.

Presently preferred R₂ groups include benzyl, n-butyl, iso-butyl,n-hexyl, ethoxyphenylpropyl, preferably 4-ethoxyphenylpropy,l andcyclopentylmethyl. Of these, isobutyl and ethoxyphenylpropyl,particularly 4-ethoxyphenylpropyl, are presently more preferred.

The group R₃

-   R₃ may for example be C₁-C₆ alkyl, phenyl, 2,-3-, or 4-pyridyl, 2-    or 3-thienyl, 2,-3-, or 4-hydroxyphenyl, 2,-3-, or 4-methoxyphenyl,    2,-3-, or 4-pyridylmethyl, benzyl, 2,-3-, or 4-hydroxybenzyl, 2,-3-,    or 4-benzyloxybenzyl, 2,-3-, or 4-C₁-C₆ alkoxybenzyl, or    benzyloxy(C₁-C₆alkyl)-; or-   the characterising group of a natural α-amino acid, in which any    functional group may be protected, any amino group may be acylated    and any carboxyl group present may be amidated; or    -   a group -[Alk]_(n)R₆ where Alk is a (C₁-C₆)alkyl or        (C₂-C₆)alkenyl group optionally interrupted by one or more —O—,        or —S— atoms or —N(R₇)— groups [where R₇ is a hydrogen atom or a        (C₁-C₆)alkyl group], n is 0 or 1, and R₆ is an optionally        substituted cycloalkyl or cycloalkenyl group; or    -   a benzyl group substituted in the phenyl ring by a group of        formula —OCH₂COR₈ where R₈ is hydroxyl, amino, (C₁-C₆)alkoxy,        phenyl(C₁-C₆)alkoxy, (C₁-C₆)alkylamino, di((C₁-C₆)alkyl)amino,        phenyl(C₁-C₆)alkylamino, the residue of an amino acid or acid        halide, ester or amide derivative thereof, said residue being        linked via an amide bond, said amino acid being selected from        glycine, □ or □ alanine, valine, leucine, isoleucine,        phenylalanine, tyrosine, tryptophan, serine, threonine,        cysteine, methionine, asparagine, glutamine, lysine, histidine,        arginine, glutamic acid, and aspartic acid; or    -   a heterocyclic(C₁-C₆)alkyl group, either being unsubstituted or        mono- or di-substituted in the heterocyclic ring with halo,        nitro, carboxy, (C₁-C₆)alkoxy, cyano, (C₁-C₆)alkanoyl,        trifluoromethyl (C₁-C₆)alkyl, hydroxy, formyl, amino,        (C₁-C₆)alkylamino, di-(C₁-C₆)alkylamino, mercapto,        (C₁-C₆)alkylthio, hydroxy(C₁-C₆)alkyl, mercapto(C₁-C₆)alkyl or        (C₁-C₆)alkylphenylmethyl; or        a group —CR_(a)R_(b)R_(c) in which:    -   each of R_(a), R_(b) and R_(c) is independently hydrogen,        (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,        phenyl(C₁-C₆)alkyl, (C₃-C₈)cycloalkyl; or    -   R_(c) is hydrogen and R_(a) and R_(b) are independently phenyl        or heteroaryl such as pyridyl; or    -   R_(c) is hydrogen, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,        phenyl(C₁-C₆)alkyl, or (C₃-C₈)cycloalkyl, and R_(a) and R_(b)        together with the carbon atom to which they are attached form a        3 to 8 membered cycloalkyl or a 5- to 6-membered heterocyclic        ring; or    -   R_(a), R_(b) and R_(c) together with the carbon atom to which        they are attached form a tricyclic ring (for example adamantyl);        or    -   R_(a) and R_(b) are each independently (C₁-C₆)alkyl,        (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, phenyl(C₁-C₆)alkyl, or a group        as defined for R_(c) below other than hydrogen, or R_(a) and        R_(b) together with the carbon atom to which they are attached        form a cycloalkyl or heterocyclic ring, and R_(c) is hydrogen,        —OH, —SH, halogen, —CN, —CO₂H, (C₁-C₄)perfluoroalkyl, —CH₂OH,        —CO₂(C₁-C₆)alkyl, —O(C₁-C₆)alkyl, —O(C₂-C₆)alkenyl,        —S(C₁-C₆)alkyl, —SO(C₁-C₆)alkyl, —SO₂(C₁-C₆) alkyl,        —S(C₂-C₆)alkenyl, —SO(C₂-C₆)alkenyl, —SO₂(C₂-C₆)alkenyl or a        group -Q-W wherein Q represents a bond or —O—, —S—, —SO— or        —SO₂— and W represents a phenyl, phenylalkyl, (C₃-C₈)cycloalkyl,        (C₃-C₈)cycloalkylalkyl, (C₄-C₈)Cycloalkenyl,        (C₄-C₈)cycloalkenylalkyl, heteroaryl or heteroarylalkyl group,        which group W may optionally be substituted by one or more        substituents independently selected from, hydroxyl, halogen,        —CN, —CO₂H, —CO₂(C₁-C₆)alkyl, —CONH₂, —CONH(C₁-C₆)alkyl,        —CONH(C₁-C₆alkyl)₂, —CHO, —CH₂OH, (C₁-C₄)perfluoroalkyl,        —O(C₁-C₆)alkyl, —S(C₁-C₆)alkyl, —SO(C₁-C₆)alkyl,        —SO₂(C₁-C₆)alkyl, —NO₂, —NH₂, —NH(C₁-C₆)alkyl,        —N((C₁-C₆)alkyl)₂, —NHCO(C₁-C₆)alkyl, (C₁-C₆)alkyl,        (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₃-C₈)cycloalkyl,        (C₄-C₈)cycloalkenyl, phenyl or benzyl.

Examples of particular R₃ groups include benzyl, phenyl,cyclohexylmethyl, pyridin-3-ylmethyl, tert-butoxymethyl, iso-propyl,iso-butyl, sec-butyl, tert-butyl, 1-benzylthio-1-methylethyl,1-methylthio-1-methylethyl, and 1-mercapto-1-methylethyl.

Presently preferred R₃ groups include phenyl, benzyl, tert-butoxymethyl,iso-propyl, tert-butyl, and iso-butyl. Of these, tert-butyl and benzylare presently more preferred.

The group R₄

R₄ may be, for example, (C₁-C₆)alkyl such as methyl, ethyl, n- oriso-propyl, prop-2-yl, and tert-butyl; (C₃-C₈)cycloalkyl such ascyclopropyl or cyclopentyl; phenyl; phenyl(C₁-C₆alkyl)-such as benzyl;heteroaryl(C₁-C₆alkyl)-such as thienylmethyl; monocyclic heterocyclicsuch as morpholino; or monocyclic heteroaryl such as thienyl or furanyl.Any of the foregoing may optionally be substituted, for example bymethyl, trifluoromethyl, hydroxy, mercapto, amino or carboxy.

As mentioned above, the present compounds are useful in human orveterinary medicine since they are active as inhibitors of MMPs.Accordingly in another aspect, this invention concerns:

(i) a method of management (by which is meant treatment or prophylaxis)of diseases or conditions mediated by MMPs in mammals, in particular inhumans, which method comprises administering to the mammal an effectiveamount of a compound which is a member of the group defined above, or apharmaceutically acceptable salt thereof; and(ii) a compound which is a member of the group defined above, for use inhuman or veterinary medicine, particularly in the management (by whichis meant treatment or prophylaxis) of diseases or conditions mediated byMMP; and(iii) the use of a compound which is a member of the group defined abovein the preparation of an agent for the management (by which is meanttreatment or prophylaxis) of diseases or conditions mediated by MMPs.

Diseases or conditions mediated by MMPs include those involving tissuebreakdown such as bone resorption, inflammatory diseases, dermatologicalconditions and tumour growth or invasion by secondary metastases; inparticular rheumatoid arthritis, osteoarthritis, periodontitis,gingivitis, corneal ulceration, neuroinflammatory disorders, includingthose involving myelin degradation, for example multiple sclerosis;restenosis, emphysemia, bronchitis and asthma.

In a further aspect of the invention there is provided a pharmaceuticalor veterinary composition comprising a compound which is a member of thegroup defined above together with a pharmaceutically or veterinarilyacceptable excipient or carrier.

It will be understood that the specific dose level for any particularpatient will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,sex, diet, time of administration, route of administration, rate ofexcretion, drug combination and the severity of the particular diseaseundergoing therapy. Optimum dose levels and frequency of dosing will bedetermined by clinical trial.

The compounds with which the invention is concerned may be prepared foradministration by any route consistent with their pharmacokineticproperties. The orally administrable compositions may be in the form oftablets, capsules, powders, granules, lozenges, liquid or gelpreparations, such as oral, topical, or sterile parenteral solutions orsuspensions. Tablets and capsules for oral administration may be in unitdose presentation form, and may contain conventional excipients such asbinding agents, for example syrup, acacia, gelatin, sorbitol,tragacanth, or polyvinyl-pyrrolidone; fillers for example lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine; tablettinglubricant, for example magnesium stearate, talc, polyethylene glycol orsilica; disintegrants for example potato starch, or acceptable wettingagents such as sodium lauryl sulphate. The tablets may be coatedaccording to methods well known in normal pharmaceutical practice. Oralliquid preparations may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, for example sorbitol,syrup, methyl cellulose, glucose syrup, gelatin hydrogenated ediblefats; emulsifying agents, for example lecithin, sorbitan monooleate, oracacia; non-aqueous vehicles (which may include edible oils), forexample almond oil, fractionated coconut oil, oily esters such asglycerine, propylene glycol, or ethyl alcohol; preservatives, forexample methyl or propyl p-hydroxybenzoate or sorbic acid, and ifdesired conventional flavouring or colouring agents. For topicalapplication to the skin, the drug may be made up into a cream, lotion orointment. Cream or ointment formulations which may be used for the drugare conventional formulations well known in the art, for example asdescribed in standard textbooks of pharmaceutics such as the BritishPharmacopoeia.

For topical application to the eye, the drug may be made up into asolution or suspension in a suitable sterile aqueous or non aqueousvehicle. Additives, for instance buffers such as sodium metabisulphiteor disodium edeate; preservatives including bactericidal and fungicidalagents such as phenyl mercuric acetate or nitrate, benzalkonium chlorideor chlorhexidine, and thickening agents such as hypromellose may also beincluded.

The active ingredient may also be administered parenterally in a sterilemedium. Depending on the vehicle and concentration used, the drug caneither be suspended or dissolved in the vehicle. Advantageously,adjuvants such as a local anaesthetic, preservative and buffering agentscan be dissolved in the vehicle.

Compounds according to the present invention in which W is a hydroxamicacid group HONH(C═O)— may be prepared from corresponding compounds ofthe invention in which W is a carboxyl group —COOH or from thecorresponding protected hydroxamic acid derivatives. That process, whichforms another aspect of the invention, comprises causing an acid ofgeneral formula (IIA) or (IIB)

or an activated derivative thereof to react with hydroxylamine,O-protected hydroxylamine, or an N,O-diprotected hydroxylamine, or asalt thereof, X, R₁, R₂, R₃, and R₄ being as defined in general formula(IA) or (IB) except that any substituents in R₁, R₂, R₃, and R₄ whichare potentially reactive with hydroxylamine, O-protected hydroxylamine,the N,O-diprotected hydroxylamine or their salts may themselves beprotected from such reaction, then removing any protecting groups fromthe resultant hydroxamic acid moiety and from any protected substituentsin R₁, R₂, R₃, and R₄.

Conversion of (IIA) or (IIB) to an activated derivative such as thepentafluorophenyl, hydroxysuccinyl, or hydroxybenzotriazolyl ester maybe effected by reaction with the appropriate alcohol in the presence ofa dehydrating agent such as dicyclohexyl dicarbodiimide (DCC),N,N-dimethylaminopropyl-N-ethyl carbodiimide (EDC), or2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ).

Protecting groups as referred to above are well known per se, forexample from the techniques of peptide chemistry. Amino groups are oftenprotectable by benzyloxycarbonyl, t-butoxycarbonyl or acetyl groups, orin the form of a phthalimido group. Hydroxy groups are often protectableas readily cleavable ethers such as the t-butyl or benzyl ether, or asreadily cleavable esters such as the acetate. Carboxy groups are oftenprotectable as readily cleavable esters, such as the t-butyl or benzylester.

Examples of O-protected hydroxylamines for use in method (a) aboveinclude O-benzylhydroxylamine, O-4-methoxybenzylhydroxylamine,O-trimethylsilylhydroxylamine, and O-tert-butoxycarbonylhydroxylamine.Examples of O,N-diprotected hydroxylamines for use in method (a) aboveinclude N,O-bis(benzyl)hydroxylamine, N,O-bis(4-methoxybenzyl)hydroxylamine,N-tert-butoxycarbonyl-O-tert-butyldimethylsilylhydroxylamine,N-tert-butoxycarbonyl-O-tetrahydropyranylhydroxylamine, andN,O-bis(tert-butoxycarbonyl)hydroxylamine.

Compounds of the invention wherein W is an N-formylhydroxylamino groupH(C═O)NH(OH)— may be prepared by N-formylation of the correspondingO-protected compound in which W is —NH(OH), then removal of theO-protecting group.

Compounds according to the present invention in which W is a carboxylicacid group —COOH, ie compounds of formula (IIA) or (IIB) above, may beprepared by a process comprising: coupling an acid of formula (III) oran activated derivative thereof

with an amine of formula (IVA) or (IVB)

wherein X, R₁ R₂, R₃, and R₄ are as defined in general formula (IA) and(IB) except that any substituents in R₁, R₂, R₃, and R₄ which arepotentially reactive in the coupling reaction may themselves beprotected from such reaction, and R₁₁, represents a hydroxy protectinggroup, and subsequently removing the protecting group R₂₁, and anyprotecting groups from R₁, R₂, R₃, and R₄.

Active derivatives of acids (III) include activated esters such as thepentafluorophenyl ester, acid anhydrides and acid halides, eg chlorides.Suitable hydroxy protecting groups may be selected from those known inthe art.

Compounds of formula (IVA) and (IVB) may be prepared by methodsanalogous to the general methods for oxadiazole ring formationillustrated in Schemes 1 and 2 in Examples 1 and 2 below.

The following preparative Examples describe the preparation of compoundsuseful in accordance with the invention.

The following abbreviations have been used in the examples

-   -   DCM—Dichloromethane    -   DMF—N,N-Dimethylformamide    -   HOBT—1-Hydroxybenzotriazole    -   Pfp—Pentafluorophenol    -   WSCDI—N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide        hydrochloride    -   HCl—Hydrochloric acid    -   THF—Tetrahydrofuran    -   TFA—Trifluoroacetic acid    -   P(O-Tol)₃— Tri-O-tolylphosphine    -   AcOEt—Ethyl acetate    -   CH₃CN—Acetonitrile

Example 1 3R-[2,2-Dimethyl-1S-(5-phenyl-[1,2,4]oxadiazol-3-yl)-propylcarbamoyl]-2S-hydroxy-5-methyl-HexanohydroxamicAcid

Example 1 was prepared as outlined in Scheme 1 using proceduresdescribed below.

Step A. (1S-carbamoyl-2,2-dimethyl-propyl)-carbamic Acid Benzyl Ester

N-benzyloxycarbonyl-L-tert-butylglycine (50 g, 189 mmol) was dissolvedin DMF (500 mL) and cooled in an ice-water bath before addition of HOBT(28.05 g, 208 mmol) and WSCDI (39.8 g, 208 mmol). Reaction was stirredat 0° C. for 1 hour before addition of 0.880 ammonia solution (21 ml,377 mmol). The reaction was allowed to warm to room temperature andstirred for 18 hours. DMF was removed under reduced pressure and theresidue partitioned between ethyl acetate and 1M HCl. The organic layerwas separated and washed with 1M HCl, saturated aqueous sodiumbicarbonate solution and brine before drying over magnesium sulphate,filtration and concentration under reduced pressure to yield(1S-carbamolyl-2,2-dimethyl-propyl)-carbamic acid benzyl ester as awhite solid (44.1 g, 89%).

¹H-NMR; delta (CDCl3), 7.32 (5H, m), 6.05 (1H, bs), 5.71 (1H, bs), 5.60(1H, d, J=6.5 Hz), 5.08 (2H, s), 4.01 (1H, d, J=6.5 Hz) and 1.00 (9H,s).

LRMS; +ve ion 265 (M+H), 287 (M+Na).

Step B. (1S-cyano-2,2-dimethyl-propyl)-carbamic Acid Benzyl Ester

(1S-carbamolyl-2,2-dimethyl-propyl)-carbamic acid benzyl ester (44.1 g,167 mmol) was dissolved in anhydrous pyridine (203 ml, 2.5 mol) under aninert atmosphere and cooled in an ice-water bath. Phosphorus oxychloride(21.8 ml, 234 mmol) was added slowly over 15 minutes and the reactionallowed to stir in the ice-water bath for 2 hours before warming to roomtemperature and stirred for 12 hours. The reaction mixture was treatedwith ice-water (400 ml) and extracted with ethyl acetate (2×300 ml). Theorganic layer was separated and washed with 1M HCl, saturated aqueoussodium bicarbonate solution and brine before drying over magnesiumsulphate, filtration and concentration under reduced pressure. Columnchromatography on silica gel using ethyl acetate/hexane as eluent leadsto isolation of the desired product as an orange oil (36.72 g, 89%).

¹H-NMR; delta (CDCl₃), 7.42 (5H, m), 5.28 (2H, m), 4.55 (2H, d, J=6.5Hz) and 1.11 (9H, s),

LRMS; +ve ion 269 (M+Na), 247.2 (M+H),

Step C. [1S—(N-hydroxycarbamimidoyl)-2,2-dimethyl-propyl]-carbamic AcidBenzyl Ester

(1S-cyano-2,2-dimethyl-propyl)-carbamic acid benzyl ester (37.60 g, 153mmol) was dissolved in ethanol (300 ml) and treated dropwise with 50%aqueous hydroxylamine (51 ml, 764 mmol). The reaction was heated toreflux and stirred for 3 hours. The reaction was then cooled andconcentrated under reduced pressure to yield the desired product as awhite foam/gum (41.5 g, 97%).

¹H-NMR; delta (CDCl₃), 7.32 (5H, m), 6.21 (1H, bs), 5.95 (1H, bs), 5.81(1H, d, J=6.4 Hz), 5.08 (2H, m), 4.79 (1H, bs), 4.05 (1H, d, J=6.5 Hz)and 0.95 (9H, s).

LRMS; +ve ion 279.8 (M+H).

Step D.[2,2-dimethyl-1S-(5-phenyl-[1,2,4]oxadiazol-3-yl)-propyl]-carbamic AcidBenzyl Ester

[1S—(N-hydroxycarbamimidoyl)-2,2-dimethyl-propyl]-carbamic acid benzylester (0.21 g, 0.75 mmol) was dissolved in DMF (5 mL) and treated withpyridine (0.1 ml, 1.28 mmol), benzoyl chloride (0.13 ml, 1.1 mmol) andDMAP (catalytic). The reaction mixture was stirred at room temperaturefor 4 hour before heating to 100° C. and stirring for 16 hours. Thereaction was cooled back to room temperature and concentrated underreduced pressure. The reaction was diluted with ethyl acetate and washedwith 1M HCl, saturated aqueous sodium bicarbonate solution and brinebefore drying over magnesium sulphate, filtration and concentrationunder reduced pressure. The desired product was isolated as an orangeoil (0.22 g, 78%).

¹H-NMR; delta (CDCl3), 8.12 (2H, m), 7.55 (3H, m), 7.32 (5H, m), 5.55(1H, d, J=6.4 Hz), 5.12 (2H, m), 4.95 (1H, d, J=6.5 Hz) and 1.10 (9H,s).

LRMS; +ve ion 366.2 (M+H), 388.2 (M+Na).

Step E. 2,2-dimethyl-1S-(5-phenyl-[1,2,4]oxadiazol-3-yl)-propylamine

[2,2-dimethyl-1S-(5-phenyl-[1,2,4]oxadiazol-3-yl)-propyl]-carbamic acidbenzyl ester (0.2 g, 0.5 mmol) was treated with 48% hydrobromic acid inacetic acid (10 ml). The reaction mixture was stirred at roomtemperature for 3 hours. The reaction was concentrated under reducedpressure and partitioned between ethyl acetate and 1M Na₂CO₃. Theorganic layer was further washed with 1M Na₂CO₃ and brine before dryingover magnesium sulphate, filtration and concentration under reducedpressure. The product was isolated as a yellow oil (0.13 g, 98%).

LRMS; +ve ion 232 (M+H).

Step F. 2R-(2,2-Dimethyl-5-oxo-[1,3]dioxolan-4S-yl)-4-methyl-pentanoicAcid

[2,2-dimethyl-1S-(5-phenyl-[1,2,4]oxadiazol-3-yl)-propyl]-amide

2,2-dimethyl-1S-(5-phenyl-[1,2,4]oxadiazol-3-yl)-propylamine (0.13 g,0.6 mmol) was dissolved in DMF (5 ml) and cooled in an ice-water bathbefore the addition2R-(2,2-Dimethyl-5S-oxo-[1,3]dioxolan-4-yl)-4-methyl-pentanoic acidpentafluorophenyl ester (0.22 g, 0.6 mmol). Reaction was allowed to warmto room temperature and stirred for 15 hours. The DMF was removed underreduced pressure and the reaction diluted with ethyl acetate and washedwith 1M HCl, saturated aqueous sodium bicarbonate solution and brinebefore drying over magnesium sulphate, filtration and concentrationunder reduced pressure. Column chromatography on silica gel using ethylacetate and hexane (1:1) lead to isolation of the desired product as awhite solid (0.16 g, 64%).

1H-NMR; delta (CDCl3), 8.12 (2H, m), 7.55 (3H, m), 6.65 (1H, d, J=6.4Hz), 5.25 (1H, d, J=6.5 Hz), 4.55 (1H, d, J=5.9 Hz), 2.75 (1H, m), 1.64(3H, s), 1.55 (3H, s), 1.04 (9H, s) and 0.88 (6H, m).

LRMS; +ve ion 444 (M+H).

Step G.3R-[2,2-Dimethyl-1S-(5-phenyl-[1,2,4]oxadiazol-3-yl)-propylcarbamoyl]-2S-hydroxy-5-methyl-hexanohydroxamicAcid

2R-(2,2-Dimethyl-5S-oxo-[1,3]dioxolan-4-yl)-4-methyl-pentanoic acid[2,2-dimethyl-1S-(5-phenyl-[1,2,4]oxadiazol-3-yl)-propyl]-amide (0.05 g,0.11 mmol) was dissolved in methanol (2 ml) and treated with 50% aqueoushydroxylamine (0.04 ml, 0.5 mmol). Reaction was stirred at roomtemperature for 2 hours before evaporation under reduced pressure. Thereaction product was separated by preparative reverse phasechromatography to yield the required product as a white solid (0.02 g,44%).

¹H-NMR; delta (CH3OD), 8.13 (2H, m), 7.65 (1H, m), 7.58 (2H, m), 5.14(1H, s), 4.01 (1H, d, J=7.1 Hz), 2.94 (1H, m), 1.60 (1H, m), 1.45 (1H,m), 1.16 (1H, m), 1.07 (9H, s), 0.89 (3H, d, J=6.5 Hz) and 0.86 (3H, d,J=6.6 Hz).

¹³C-NMR; delta (CH3OD), 177.1, 176.3, 172.0, 171.6, 134.6, 130.8, 129.4,125.7, 73.7, 55.8, 49.6, 39.7, 36.2, 27.4, 27.2, 24.2 and 22.5.

LRMS; +ve ion 419 (M+H); −ve ion 417 (M−H).

Step H. 2R-(2,2-Dimethyl-5-oxo-[1,3]dioxolan-4S-yl)-4-methyl-pentanoicAcid Pentafluorophenyl Ester

2R-(2,2-Dimethyl-5-oxo-[1,3]dioxolan-4S-yl)-4-methyl-pentanoic acid(prepared according to WO 94/02447) (30 g, 130 mmol) was dissolved inethyl acetate (300 ml) and treated with pentafluorophenol (28.8 g, 156mmol) and WSCDI (30 g, 156 mmol). Reaction was heated to reflux for 2hours and then allowed to stir at room temperature for 12 hours. Thereaction mixture was washed with 1M Na₂CO₃ and brine before drying overmagnesium sulphate, filtration and concentration under reduced pressure.The product was recrystallised from ethyl acetate/hexane to yield thedesired product as a single diastereomer (21.2 g, 42%).

¹H-NMR; delta (CDCl3), 4.55 (1H, d, J=6.7 Hz), 3.31 (1H, m), 1.85 (3H,bm), 1.65 (3H, s), 1.58 (3H, s), 1.05 (3H, d, J=6.5 Hz) and 0.99 (3H, d,J=6.5 Hz).

Also prepared, the diastereomer3R-[2,2-dimethyl-1S-(5-phenyl-[1,2,4]oxadiazol-3-yl)-propylcarbamoyl]-2R-hydroxy-5-methyl-hexanohydroxamicacid.

M+H=420.0, M+Na=441.5, M−H=417.5.

The corresponding carboxylic acid was prepared as outlined in Scheme 1and the procedure below.

Step I.3R-[1S-(5-Furan-2-yl-[1,2,4]oxadiazol-3-yl)-2,2-dimethyl-propylcarbamoyl]-2S-hydroxy-5-methyl-hexanoicAcid

2R-(2,2-Dimethyl-5S-oxo-[1,3]dioxolan-4-yl)-4-methyl-pentanoic acid[2,2-dimethyl-1S-(5-Furan-2-yl-[1,2,4]oxadiazol-3-yl)-propyl]-amide(0.05 g, 0.12 mmol) was dissolved in tetrahydrofuran (5 ml) and cooledto 4° C. during the addition of 1M hydrochloric acid (5 ml). Thesolution was allowed to warm to room temperature and then stirred for 18hours. The bulk of the solvent was removed under reduced pressure beforedrying under high vacuum to a white foam (0.045 g, ca. quant.).

1H-NMR; delta (CH3OD), 7.88 (1H, s), 7.45 (1H, d, J=3.6 Hz), 6.74 (1H,m), 5.15 (1H, s), 4.18 (2H, d, J=6.4 Hz), 2.91 (1H, m), 1.65 (1H, m),1.50 (1H, m), 1.31 (1H, m), 1.06 (9H, s), 0.88 (3H, d, J=6.4 Hz) and0.82 (3H, d, J=6.5 Hz).

LRMS; −ve ion 392.2 (M−H).

Example 23R-[2,2-Dimethyl-1S-(3-phenyl-[1,2,4]oxadiazol-5-yl)-propylcarbamoyl]-2S-hydroxy-5-methyl-hexanohydroxamicAcid

Example 2 was prepared as outlined in scheme 2 using proceduresdescribed below.

Step A. 2S-tert-Butoxycarbonylamino-3,3-dimethyl-butyric AcidBenzotriazol-1-yl Ester

A solution of N-tert-Butoxycarbonyl-L-tert-butyl glycine (5 g, 21.6mmol) in ethyl acetate (80 mL) was cooled in an ice-water bath. HOBT(3.22 g, 23.8 mmol) and WSCDI (4.56 g, 23.8 mmol) were added and thereaction allowed to stir at room temperature for 12 hours. The reactionmixture was washed with 1M Na₂CO₃ and brine, before drying overmagnesium sulphate, filtration and concentration to a white foam (5.74g, 76%).

1H-NMR; delta (CDCl3), 8.05 (1H, m), 7.65 (2H, m), 7.41 (1H, m), 5.10(1H, d, J=6.7 Hz), 4.45 (1H, d, J=6.5 Hz), 1.55 (9H, s) and 1.21 (9H,s).

LRMS; +ve ion 349 (M+H).

Step B.[2,2-Dimethyl-1S-(3-phenyl-[1,2,4]oxadiazol-5-yl)-propyl]-carbamic AcidTert-butyl Ester

2S-tert-Butoxycarbonylamino-3,3-dimethyl-butyric acid benzotriazol-1-ylester (3.71 g, 10.7 mmol) was dissolved in toluene (80 mL) and treatedwith N-hydroxy-benzamidine (2.9 g, 21.3 mmol). The reaction mixture wasstirred at 110° C. for 18 hours. The solution was concentrated underreduced pressure and partitioned between ethyl acetate and 1M Na₂CO₃.The organic layer was further washed with 1M Na₂CO₃ and brine beforedrying over magnesium sulphate, filtration and concentration underreduced pressure. Column chromatography on silica gel using ethylacetate and hexane (1:4) lead to isolation of the desired product (2.58g, 73%).

1H-NMR; delta (CDCl3), 8.10 (2H, m), 7.50 (3H, m), 5.30 (1H, bd), 4.95(1H, d, J=6.5 Hz), 1.44 (9H, s) and 1.03 (9H, s).

LRMS; +ve ion 354.2 (M+Na).

Step C. N-hydroxy-benzamidine

Benzonitrile (5 g, 48 mmol) was dissolved in ethanol (100 ml) andtreated with 50% aqueous hydroxylamine (16 ml, 242 mmol). Reaction washeated to reflux for 3 hours before concentration under reduced pressureto give a clear foam (4.5 g, 68%).

LRMS; +ve ion 137 (M+H).

Step D. 2,2-Dimethyl-1S-(3-phenyl-[1,2,4]oxadiazol-5-yl)-propylamine

[2,2-Dimethyl-1S-(3-phenyl-[1,2,4]oxadiazol-5-yl)-propyl]-carbamic acidtert-butyl ester (1 g, 3.0 mmol) was dissolved in DCM (5 ml) and treatedwith TFA (5 ml). Reaction stirred at room temperature for 3 hours. Thereaction was concentrated under reduced pressure and partitioned betweenethyl acetate and 1M Na₂CO₃. The organic layer was further washed with1M Na₂CO₃ and brine before drying over magnesium sulphate, filtrationand concentration under reduced pressure to give the desired product(0.65 g, 93%).

¹H-NMR; delta (CH3OD), 8.10 (2H, m), 7.55 (3H, m), 4.81 (1H, s) and 1.19(9H, s).

LRMS; +ve ion 232 (M+H).

Step E. 2R-(2,2-Dimethyl-5-oxo-[1,3]dioxolan-4S-yl)-4-methyl-pentanoicacid-[2,2-dimethyl-1S-(3-phenyl-[1,2,4]oxadiazol-5-yl)-propyl]-amide

2R-(2,2-Dimethyl-5-oxo-[1,3]dioxolan-4S-yl)-4-methyl-pentanoic acid(0.27 g, 1.17 mmol) was dissolved in DMF (5 ml) and cooled in anice-water bath before addition of HOBT (0.17 g, 1.29 mmol) and WSCDI(0.25 g, 1.29 mmol). Reaction was stirred at 0° C. for 1 hour beforeaddition of 2,2-Dimethyl-1S-(3-phenyl-[1,2,4]oxadiazol-5-yl)-propylamine(0.3 g, 1.29 mmol). The reaction was allowed to warm to room temperatureand stirred for 18 hours. DMF was removed under reduced pressure and theresidue partitioned between ethyl acetate and 1M HCl. The organic layerwas separated and washed with 1M HCl, saturated aqueous sodiumbicarbonate solution and brine before drying over magnesium sulphate,filtration and concentration under reduced pressure. Columnchromatography on silica gel using ethyl acetate and hexane (1:4) leadto isolation of the desired product (0.26 g, 46%).

¹H-NMR; delta (CDCl3), 8.10 (2H, m), 7.50 (3H, m), 6.80 (1H, d, J=9.3Hz), 5.24 (1H, d, J=9.3 Hz), 4.55 (1H, d, J=5.1 Hz), 2.81 (1H, m), 1.63(3H, s), 1.55 (3H, s), 0.92 (3H, d, J=6.1 Hz) and 0.89 (3H, d, J=6.2Hz).

LRMS; +ve ion 444 (M+H).

Step F.3R-[2,2-Dimethyl-1S-(3-phenyl-[1,2,4]oxadiazol-5-yl)-propylcarbamoyl]-2S-hydroxy-5-methyl-hexanohydroxamicAcid

2R-(2,2-Dimethyl-5-oxo-[1,3]dioxolan-4S-yl)-4-methyl-pentanoic acid[2,2-dimethyl-1S-(3-phenyl-[1,2,4]oxadiazol-5-yl)-propyl]-amide (0.26 g,0.6 mmol) was dissolved in methanol (5 ml) and treated with 50% aqueoushydroxylamine (0.2 ml, 2.95 mmol). Reaction stirred at room temperaturefor 3 hrs before concentration under reduced pressure. The product wasrecrystallised from ethyl acetate/hexane to yield the desired product(0.11 g, 41%).

¹H-NMR; delta(CH3OD), 8.06 (2H, m), 7.53 (3H, m), 5.21 (1H, s), 4.01(1H, d, J=7.5 Hz), 2.99 (1H, m), 1.60 (1H, m), 1.50 (1H, m), 1.15 (1H,m), 1.10 (9H, s), 0.92 (3H, d, J=6.6 Hz) and 0.81 (3H, d, J=6.5 Hz).

¹³C-NMR; delta (CH3OD), 180.3, 176.7, 172.0, 169.7, 132.9, 130.5, 128.8,128.4, 73.7, 57.1, 49.5, 39.5, 36.5, 27.3, 24.3 and 22.5.

LRMS; +ve ion 419 (M+H); −ve ion 417 (M−1).

Example 32R-[3-(4-Ethoxy-phenyl)-propyl]-N₁-[1S-(5-thiophen-2-yl)-[1,2,4]oxadiazol-3-yl)-2,2-dimethyl-propyl]-3S,N₄-dihydroxy-succinamide

Example 3 was prepared as outlined in Scheme 3 using proceduresdescribed below.

Step A: 2R-allyl-3S-hydroxy-succinic Acid Diisopropylester

To a cold (−78 C) solution of 2S-Hydroxy-succinic acid diisopropyl ester(19.70 ml, 95 mmol) in THF (35 ml) was added LiHMDS (200 ml, 0.2 mol,2.1 eq.) dropwise. The reaction mixture was stirred at −78 C for twohours and then at −30 C for 30 min. The reaction mixture was then cooledto −78 C and allyl bromide (12.36 ml, 0.14 mol. 1.5 eq.) was addeddropwise. The reaction mixture was allowed to warm to RT overnight. Itwas poured into a saturated solution of NH₄Cl/ice (200 ml). Extractionwith AcOEt (3×200 ml) followed by a wash with water (50 ml) and withbrine (50 ml) afforded a yellow oil after removal of the solvents undervacuum. Purification by flash chromatography gave2R-allyl-3S-hydroxy-succinic acid diisopropylester as a colourless oil(7.76 g, de=80%, 40% yield).

¹H-NMR; delta (CDCl₃), 5.77-5.88 (1H, m), 4.98-5.21 (4H, m), 4.22 (1H,brs), 3.18 (1H, bs), 2.87-2.94 (1H, m), 2.56-2.65 (1H, m), 2.40-2.48(1H, m), 1.29 (6H, d, J=6.3 Hz) and 1.22 (6H, d, J=6.3 Hz).

LRMS: +ve ion 281 (M+Na).

Step B: 2R-[3-(4-ethoxy-phenyl)-allyl]-3S-hydroxy-succinic aciddiisopropyl ester

To a solution of 2R-allyl-3S-hydroxy-succinic acid diisopropylester(4.79 g, 18.5 mmol), 4-bromo phenetole (3.19 ml, 22.2 mmol, 1.2 eq.) andNEt₃ (6.22 ml, 44.6 mmol, 2.4 eq.) in CH₃CN (40 ml), was added asonicated (for 2 min) suspension of P(O-Tol)₃ (0.57 g, 2.22 mmol, 0.1eq.) and Pd(OAc)₂ (209 mg, 5%) in CH₃CN (5 ml). The reaction mixture washeated to reflux for 2 hrs. CH₃CN was removed under vacuum. The crudewas extracted with AcOEt (3×200 ml), washed with water (50 ml) and withbrine (50 ml). A purification by flash chromatography afforded thedesired 2R-[3-(4-ethoxy-phenyl)-allyl]-3S-hydroxy-succinic aciddiisopropyl ester (5.92 g, 84% yield).

1H-NMR; delta (CDCl₃), 7.28 (2H, d, J=8.8 Hz), 6.83 (2H, d, J=8.8 Hz),6.46 (1H, d, J=15.7 Hz), 6.02-6.12 (1H, m), 4.98-5.13 (2H, m), 4.26 (1H,dd, J=7.1, 3.0 Hz), 4.02 (2H, q, J=7.0 Hz), 3.23 (1H, d, J=7.1 Hz),2.92-2.97 (1H, m), 2.68-2.79 (1H, m), 2.49-2.62 (1H, m), 1.41 (3H, t,J=7.0 Hz) and 1.19-1.30 (12H, m).

LRMS: +ve ion 400 (M+Na).

Step C: 2R-[3-(4-ethoxy-phenyl)-propyl]-3S-hydroxy-succinic AcidDiisopropyl Ester

To a solution of 2R-[3-(4-ethoxy-phenyl)-allyl]-3S-hydroxy-succinic aciddiisopropyl ester (129 mg, 0.34 mmol) in MeOH (10 ml) under an inertatmosphere, was added 10% Pd/C (13 mg). H₂ was bubbled through theresulting suspension for 30 min. The reaction mixture was then stirredunder 1 atmosphere of H₂ for 16 hrs. Pd/C was filtered off and thesolvent removed under reduced pressure to give2R-[3-(4-ethoxy-phenyl)-propyl]-3S-hydroxy-succinic acid diisopropylester (115 mg, 88% yield).

¹H-NMR; delta (CDCl₃), 7.08 (2H, d, J=8.6 Hz), 6.81 (2H, d, J=8.6 Hz),4.97-5.14 (2H, m), 4.20 (1H, dd, J=7.3, 3.5 Hz), 4.01 (2H, q, J=7.0 Hz),3.18 (1H, d, J=7.3 Hz), 2.77-2.83 (1H, m), 2.55-2.62 (2H, m), 1.45-1.94(4H, m), 1.40 (3H, t, J=7.0 Hz) and 1.12-1.30 (12H, m).

LRMS: +ve ion 402.0 (M+Na).

Step D: 2R-[3-(4-ethoxy-phenyl)-propyl]-3S-hydroxy-succinic Acid

To a solution of 2R-[3-(4-ethoxy-phenyl)-propyl]-3S-hydroxy-succinicacid diisopropyl ester (4.78 g, 12.6 mmol) in THF/water (3:1, 120 ml)was added NaOH (1.66 g, 41.5 mmol, 5.5 eq.). The reaction mixture wasthen stirred for 16 hrs at RT. The mixture was concentrated underreduced pressure and acidify to pH=3 by addition of HCl 1N. The hydroxydiacid was extracted with AcOEt. The organic layer was dried over MgSO₄and the solvent removed under reduced pressure to give the desired2R-[3-(4-ethoxy-phenyl)-propyl]-3S-hydroxy-succinic acid (3.66 g, 85%yield).

1H-NMR; delta (CH3OD), 7.07 (2H, d, J=8.6 Hz), 6.79 (2H, d, J=8.6 Hz),4.23 (1H, d, J=5.8 Hz), 3.98 (2H, q, J=7.0 Hz), 2.76-2.81 (1H, m),2.53-2.59 (2H, m), 1.55-1.72 (4H, m), 1.35 (3H, t, J=7.0 Hz).

LRMS: +ve ion 319 (M+Na); −ve ion 295 (M−H).

Step E:2R-(2,2-dimethyl-5-oxo-[1,3]dioxolan-4S-yl)-5-(4-ethoxy-phenyl)-pentanoicacid

To a solution of 2R-[3-(4-ethoxy-phenyl)-propyl]-3S-hydroxy-succinicacid (3.66 g, 12.3 mmol) in acetone (50 ml) under an inert atmospherewere added dimethoxy propane (2.58 ml, 21 mmol, 1.7 eq.) and copperchloride (165 mg, 1.2 mmol, 0.1 eq.). The reaction mixture was stirredat RT for 16 hrs. The solvent was then removed under vacuum to give2R-(2,2-dimethyl-5-oxo-[1,3]dioxolan-4S-yl)-5-(4-ethoxy-phenyl)-pentanoicacid (4.03 g, 97% yield).

¹H-NMR; delta (CDCl₃), 7.08 (2H, d, J=8.5 Hz), 6.82 (2H, d, J=8.5 Hz),4.48 (1H, d, J=4.8 Hz), 4.01 (2H, q, J=7.0 Hz), 2.91-2.98 (1H, m),2.54-2.64 (3H, m), 1.23-2.20 (4H, m), 1.58 (3H, s), 1.53 (3H, s) and1.40 (3H, t, J=7.0 Hz).

LRMS: +ve ion 359 (M+Na); −ve ion 335 (M−H).

Step F.2R-(2,2-dimethyl-5-oxo-[1,3]dioxolan-4S-yl)-5-(4-ethoxy-phenyl)-pentanoicAcid Pentafluorophenyl Ester

To a cold (0° C.) solution of2R-(2,2-dimethyl-5-oxo-[1,3]dioxolan-4S-yl)-5-(4-ethoxy-phenyl)-pentanoicacid (4.03 g, 12 mmol) and pentafluoro phenol (2.43 g, 13.2 mmol, 1.1eq.) in CH₂Cl₂ (50 ml) was added WSC (2.54 g, 13.2 mmol, 1.1 eq.). Thereaction mixture was allowed to warm to RT overnight. CH₂Cl₂ was removedunder vacuum and the resulting crude reaction mixture was dissolved inAcOEt (200 ml). The organic layer was washed with water (50 ml),NaHCO_(3 sat) (20 ml) and finally with brine (20 ml). Solvent wasremoved under reduced pressure to give an oil which was purified byflash chromatography to furnish the expected2R-(2,2-dimethyl-5-oxo-[1,3]dioxolan-4S-yl)-5-(4-ethoxy-phenyl)-pentanoicacid pentafluorophenyl ester (3.94 g, 65% yield).

1H-NMR; delta (CDCl₃), 7.09 (2H, d, J=8.4 Hz), 6.83 (2H, d, J=8.4 Hz),4.56 (1H, d, J=6.0 Hz), 4.01 (2H, q, J=7.0 Hz), 3.20-3.28 (1H, m), 2.64(2H, t, J=7.6 Hz), 1.98-2.08 (2H, m), 1.70-1.86 (2H, m), 1.62 (3H, s),1.57 (3H, s) and 1.40 (3H, t, J=7.0 Hz).

Step G.2R-[3-(4-Ethoxy-phenyl)-propyl]-N₁-[1S-(5-thiophen-2-yl)-[1,2,4]oxadiazol-3-yl)-2,2-dimethyl-propyl]-[1,3]dioxolan-4S-one

To a solution of2R-(2,2-dimethyl-5-oxo-[1,3]dioxolan-4S-yl)-5-(4-ethoxy-phenyl)-pentanoicacid pentafluorophenyl ester (150 mg, 0.30 mmol) in CH₂Cl₂ (10 ml) wasadded2,2-dimethyl-1S-(5-thiophen-2-yl)-[1,2,4]oxadiazol-3-yl)-propylamine(100 mg, 0.42 mmol, 1.4 eq.). The reaction mixture was stirred for 16hrs and the solvent was removed under vacuum. The crude was taken-up inAcOEt (70 ml) and washed with water (10 ml), then with Na₂CO₃ (10 ml)and finally with brine (10 ml). The solvent was dried over MgSO₄ andremoved under reduced pressure to give the desired2R-[3-(4-Ethoxy-phenyl)-propyl]-N₁-[1S-(5-thiophen-2-yl)-[1,2,4]oxadiazol-3-yl)-2,2-dimethyl-propyl]-[1,3]dioxolan-4S-one(82 mg, 33% crude).

¹H-NMR; delta (CDCl₃), 7.88 (1H, m), 7.62 (1H, m), 7.20 (1H, m), 6.95(2H, d, J=8.4 Hz), 6.71 (2H, d, J=8.4 Hz), 6.55 (1H, d, J=9.7 Hz), 5.19(1H, d, J=9.7 Hz), 4.56 (1H, d, J=6.4 Hz), 3.95 (2H, q, J=7.0 Hz), 2.64(3H, bm), 1.84 (2H, m), 1.70 (2H, m), 1.62 (3H, s), 1.54 (3H, s), 1.38(3H, t, J=6.9 Hz) and 1.02 (9H, s).

LRMS: +ve ion 556.0 (M+H).

Step H.2R-[3-(4-Ethoxy-phenyl)-propyl]-N₁-[1S-(5-thiophen-2-yl)-[1,2,4]oxadiazol-3-yl)-2,2-dimethyl-propyl]-3S,N₄-dihydroxy-succinamide

To a solution of2R-[3-(4-Ethoxy-phenyl)-propyl]-N₁-[1S-(5-thiophen-2-yl)-[1,2,4]oxadiazol-3-yl)-2,2-dimethyl-propyl]-[1,3]dioxolan-4S-one(82 mg, 0.15 mmol) in i-PrOH (5 ml), was added an aqueous solution ofhydroxylamine (50%, 48 μl, 0.7 mmol, 5 eq.). The reaction mixture wasallowed to stir at RT for 16 hrs. The solvent was removed under reducedpressure to yield an oil which was purified by preparative reverse phasechromatography to give the required product (25.3 mg, 32%).

¹H-NMR; delta(CH3OD), ¹H-NMR; delta(CH3OD), 7.86 (2H, m), 7.25 (1H, dd,J=3.8 Hz), 6.83 (2H, d, J=8.6 Hz), 6.54 (2H, d, J=8.6 Hz), 5.14 (1H, s),4.03 (1H, d, J=7.6 Hz), 3.87 (2H, q, J=6.96), 2.88 (1H, m), 2.45 (2H,bm), 1.53 (4H, bm), 1.33 (3H, t, J=7.0 Hz) and 1.06 (9H, s).

LRMS: +ve ion 553.2 (M+Na); −ve ion 529.2 (M−H)

The compounds of Examples 4-17 were prepared by the method of Example 1by parallel synthesis, using the appropriate acid chloride in Step D.The products were purified by preparative HPLC:

Example 42S-Hydroxy-3R-[1S-(5-isopropyl-[1,2,4]oxadiazol-3-yl)-2,2-dimethyl-propylcarbamoyl]-5-methylHexanohydroxamic Acid

LRMS; +ve ion 407 (M+Na); −ve ion 383 (M−H)

Example 52S-Hydroxy-3R-[1S-(5-furan-2-yl-[1,2,4]oxadiazol-3-yl)-2,2-dimethyl-propylcarbamoyl]-5-methylHexanohydroxamic Acid

LRMS; +ve ion 431 (M+Na), −ve ion 407 (M−H).

Example 62S-Hydroxy-3R-[1S-(5-cyclopentylmethyl-[1,2,4]oxadiazol-3-yl)-2,2-dimethyl-propylcarbamoyl]-5-methylHexanohydroxamic Acid

LRMS; +ve ion 425 (M+H), −ve ion 423 (M−H).

Example 72S-Hydroxy-3R-[1S-(5-thiopen-2-ylmethyl-[1,2,4]oxadiazol-3-yl)-2,2-dimethyl-propylcarbamoyl]-5-methylHexanohydroxamic Acid

LRMS; +ve ion 461 (M+Na), −ve ion 437 (M−H).

Example 82S-Hydroxy-3R-[1S-(5-ethyl-[1,2,4]oxadiazol-3-yl)-2,2-dimethyl-propylcarbamoyl]-5-methylHexanohydroxamic Acid

LRMS; +ve ion 393 (M+Na), −ve ion 369 (M−H).

Example 92S-Hydroxy-3R-[1S-(5-cyclopentyl-[1,2,4]oxadiazol-3-yl)-2,2-dimethyl-propylcarbamoyl]-5-methylHexanohydroxamic Acid

LRMS; +ve ion 411 (M+H), −ve ion 409 (M−H).

Example 102S-Hydroxy-3R-[1S-(5-benzyl-[1,2,4]oxadiazol-3-yl)-2,2-dimethyl-propylcarbamoyl]-5-methylHexanohydroxamic Acid

LRMS; +ve ion 433 (M+H), −ve ion 431 (M−H).

Example 112S-Hydroxy-3R-[1S-(5-isobutyl-[1,2,4]oxadiazol-3-yl)-2,2-dimethyl-propylcarbamoyl]-5-methylHexanohydroxamic Acid

LRMS; +ve ion 421 (M+Na), −ve ion 397 (M−H).

Example 122S-Hydroxy-3R-[1S-(5-tert-butyl-[1,2,4]oxadiazol-3-yl)-2,2-dimethyl-propylcarbamoyl]-5-methylHexanohydroxamic Acid

LRMS; +ve ion 421 (M+Na), −ve ion 397 (M−H).

Example 132S-Hydroxy-3R-[1S-(5-thiophen-2-yl-[1,2,4]oxadiazol-3-yl)-2,2-dimethyl-propylcarbamoyl]-5-methylHexanohydroxamic Acid

LRMS; +ve ion 425 (M+H), −ve ion 423 (M−H).

Also prepared, the diastereomer2R-hydroxy-3R-[1S-(5-thiophen-2-yl-[1,2,4]oxadiazol-3-yl)-2,2-dimethyl-propylcarbamoyl]-5-methylhexanohydroxamic acid

M+H=425.1, M+Na=447.1, M−H=423.0.

Example 142S-Hydroxy-3R-[1S-(5-(2,2-dimethyl-propyl)-[1,2,4]oxadiazol-3-yl)-2,2-dimethyl-propylcarbamoyl]-5-methylHexanohydroxamic Acid

LRMS; +ve ion 435 (M+Na), −ve ion 411 (M−H).

Example 152S-Hydroxy-3R-[1S-(5-p-tolyl-[1,2,4]oxadiazol-3-yl)-2,2-dimethyl-propylcarbamoyl]-5-methylHexanohydroxamic Acid

LRMS; +ve ion 433 (M+H), −ve ion 431 (M−H).

Example 162S-Hydroxy-3R-[1S-(5-cyclopropyl-[1,2,4]oxadiazol-3-yl)-2,2-dimethyl-propylcarbamoyl]-5-methylHexanohydroxamic Acid

LRMS; +ve ion 405 (M+Na), −ve ion 381 (M−H).

Example 172S-Hydroxy-3R-[1S-(5-methyl-[1,2,4]oxadiazol-3-yl)-2,2-dimethyl-propylcarbamoyl]-5-methylHexanohydroxamic Acid

1H-NMR; delta(CH3OD), 8.26 (1H, d, J=9.4 Hz), 5.02 (1H, d, J=9.5 Hz),4.02 (1H, d, J=6.4 Hz), 2.89 (1H, m), 2.57 (3H, s), 1.61 (1H, m), 1.44(1H, m), 1.22 (1H, m), 1.00 (9H, s)

¹³C-NMR; delta (CH3OD), 178.6, 176.1, 171.9, 170.7, 73.5, 55.6, 49.5,39.9, 36.2, 27.6, 26.6, 24.2, 22.7 and 12.4.

LRMS; +ve ion 379 (M+Na), −ve ion 355 (M−H).

The compounds of Examples 18-19 were prepared by the method of Example2, by using the appropriate nitrile in Step C and/or the appropriateamino acid residue in Step A:

Example 182S-Hydroxy-3R-[1S-(3-isopropyl-[1,2,4]oxadiazol-3-yl)-2,2-dimethyl-propylcarbamoyl]-5-methylHexanohydroxamic Acid

¹H-NMR; delta(CH3OD), 5.12 (1H, s), 3.98 (1H, d, J=7.5 Hz), 3.06 (1H,m), 2.92 (1H, m), 1.61 (1H, m), 1.43 (1H, m), 1.31 (6H, d, J=6.9 Hz),1.14 (1H, m), 1.03 (9H, s), 0.89 (3H, d, J=6.7 Hz), 0.81 (3H, d, J=6.8Hz).

¹³C-NMR; delta (CH3OD), 179.7, 176.6, 176.5, 172.0, 73.7, 56.9, 49.2,39.5, 36.5, 28.3, 27.3, 24.5, 22.3, 21.2 and 21.1.

LRMS; +ve ion 385 (M+H), −ve ion 383 (M−H).

Example 19 2S,N₁-Dihydroxy-3R-isobutyl-N₄-[2-methyl-1S-(3-phenyl-[1,2,4]oxadiazol-5-yl)-propyl]-succinamide

¹H-NMR; delta(CH3OD), 8.05 (2H, m), 7.52 (3H, m), 5.14 (1H, d, J=7:2Hz), 4.00 (1H, d, J=7.7 Hz), 2.91 (1H m), 2.36 (1H, m), 1.63 (1H, m),1.54 (1H, m), 1.16 (1H, m), 1.09 (3H, d, J=6.8 Hz), 1.00 (3H, d, J=6.8Hz), 0.95 (3H, d, J=6.3 Hz), 0.84 (3H, d, J=6.3 Hz).

¹³C-NMR; delta (CH3OD), 181.0, 176.8, 172.0, 169.9, 132.9, 130.5, 128.7,128.4, 73.7, 54.3, 49.6, 39.5, 33.3, 27.2, 24.4, 22.5, 19.8 and 19.4.

LRMS; +ve ion 427 (M+Na), −ve ion 403 (M−H).

The compounds of Examples 20-23 were prepared by the method of Example2, by using the appropriate nitrile in Step C and/or the appropriateamino acid residue in Step A. The synthesis to the appropriate chiralsuccinate in Step E is detailed within WO 94/21625.

Example 202S-Allyl-5-methyl-3R-[2-phenyl-1S-(3-phenyl-[1,2,4]oxadiazol-5-yl)-ethylcarbamoyl]-hexanohydroxamicAcid

¹H-NMR; delta (CH3OD), 9.13 (1H, d, J=8.26 Hz), 8.05 (2H, m), 7.55 (3H,m), 7.25 (5H, m), 5.66 (1H, m), 5.45 (1H, m), 4.90 (2H, m), 4.50 (1H, s)3.51 (1H, dd, J=13.92, 4.84 Hz), 3.17 (1H, dd, J=13.92, 10.90 Hz), 2.50(1H, m), 2.0 (2H, m), 1.50 (3H, m), 1.0 (3H, d, J=6.5 Hz), 0.96 (3H, d,J=6.6 Hz).

13C-NMR; delta (CH3OD), 181.0, 177.0, 172.7, 138.0, 136.5, 133, 130.8,130.6, 130.5, 130.1, 128.7, 128.7, 117.7, 48.4, 48.3, 42.1, 39.5, 36.2,27.1, 24.9 and 22.0.

Example 212S-Allyl-5-methyl-3R-[2-phenyl-1S-(3-isopropyl-[1,2,4]oxadiazol-5-yl)-ethylcarbamoyl]-hexanohydroxamicAcid

1H-NMR; delta (DMSO), 10.28 (1H, s), 8.64 (1H, d, J=6.2 Hz), 8.64 (1H,br s), 7.25 (5H, m), 5.45 (2H, m), 4.51 (1H, m), 4.30 (2H, m), 3.15 (1H,m), 2.85 (2H, m), 2.20 (1H, dt, J=10.6, 3.12 Hz), 1.70 (2H, m), 1.25(6H, d, J=6.91 Hz), 0.70 (1H, m), 0.52 (3H, d, J=6.4 Hz), 0.48 (3H, d,J=6.4 Hz).

¹³C-NMR; delta (MEOD), 179.0, 175.6, 175.5, 171.3, 136.6, 135.0, 129.2,128.6, 127.3, 116.4, 48.7, 46.9, 40.6, 38.1, 34.8, 26.9, 25.6, 23.5,20.7 and 19.9.

Example 222S-Allyl-5-methyl-3R-[2-phenyl-1S-(3-methyl-[1,2,4]oxadiazol-5-yl)-ethylcarbamoyl]-hexanohydroxamicAcid

¹H-NMR; delta (CH3OD), 8.98 (1H, d, J=8.41 Hz), 7.27 (5H, m), 5.51 (2H,m), 4.85 (2H, m), 3.41 (1H, dd, J=14.0, 5.0 Hz), 3.14 (1H, dd, J=14.0,10.97 Hz), 2.47 (1H, dt, J=11.0, 3.25 Hz), 2.16 (3H, s), 2.00 (1H, dt,J=11.40, 3.30 Hz), 1.80 (1H, m), 1.15 (1H, m), 0.98 (3H, d, J=6.6 Hz),0.92 (3H, d, J=6.6 Hz).

¹³C-NMR; delta (CH3OD), 172.62, 168.27, 133.59, 132.07, 126.34, 125.66,124.28, 113.36, 45.19, 44.04, 43.95, 37.61, 35.15, 31.75, 22.72, 20.44,17.59 and 7.36.

Example 23 2S-Allyl-3R-[2,2-dimethyl-IS-(3-methyl-[1,2,4]oxadiazol-5-yl)-propylcarbamoyl]-5-methyl-hexanohydroxamicAcid

¹H-NMR; delta (CH3OD), 8.81 (1H, d, J=8.59 Hz), 7.65 (1H, m), 5.70 (1H,m), 5.15 (1H, d, J=8.62 Hz), 4.95 (2H, m), 2.60 (1H, dt, J=11.10, 3.16Hz), 2.39 (3H, s), 1.38 (1H, dt, J=13.10, 3.33 Hz), 1.31 (1H, m), 0.98(1H, m), 0.98 (9H, s), 0.86 (3H, d, J=6.6 Hz), 0.84 (3H, d, J=6.6 Hz).

The compound of Example 24 was prepared by the method of Example 2. Thesynthesis to the appropriate chiral succinate in Step E is detailedwithin WO 95/19956

Example 243R-[2,2-Dimethyl-1S-(3-phenyl-[1,2,4]oxadiazol-5-yl)-propylcarbamoyl]-5-methyl-hexanohydroxamicAcid

LRMS; +ve ion 403.5 (M+H), −ve ion 401.3 (M−H).

The compound of Example 25 was prepared by the method of Example 2, byusing the appropriate nitrile in Step C and/or the appropriate aminoacid residue in Step A. The synthesis to the appropriate chiralsuccinate in Step E is detailed within WO 97/02239.

Example 252S-Methoxy-5-methyl-3R-[1S-(3-methyl-[1,2,4]oxadiazol-5-yl)-2-phenyl-ethylcarbamoyl]-hexanohydroxamicAcid

¹H-NMR; delta (CH3OD), 7.14 (5H, m), 5.34 (1H, m), 3.38 (1H, d, J=9.68Hz), 3.20 (2H, m), 3.02 (3H, s), 2.65 (1H, m), 2.22 (3H, s), 1.35 (2H,m), 0.90 (1H, m), 0.73 (3H, d, J=6.55 Hz), and 0.70 (3H, d, J=6.57 Hz).

The compounds of Example 26 and 27 were prepared by the method ofExample 2. The synthesis to the appropriate chiral succinate in Step Eis detailed within WO 92/13831 using methods analogous to thosedescribed in WO 95/32944.

Example 263R-[2,2-Dimethyl-1S-(3-phenyl-[1,2,4]oxadiazol-5-yl)-propylcarbamoyl]-heptadecanoicAcid

¹H-NMR; delta(CH3OD), 8.05 (2H, m), 7.49 (3H, m), 5.22 (1H, s), 2.93(1H, m), 2.65 (1H, dd, J=9.8, 16.7 Hz), 2.38 (1H, dd, J=4.6, 16.6 Hz),1.52 (1H, m), 1.43 (1H, m), 1.26 (24H, m), 1.10 (9H, s) and 0.89 (3H,m).

LRMS; +ve ion 528.4 (M+H).

Example 273R-[2,2-Dimethyl-1S-(3-phenyl-[1,2,4]oxadiazol-5-yl)-propylcarbamoyl]-nonadecanoicAcid

LRMS; +ve ion 556.2 (M+H).

The compound of Example 28 was prepared by the method of Example 1. Thesynthesis to the appropriate chiral succinate in Step H is detailedwithin WO 92/13831 using methods analogous to those described in WO95/32944.

Example 286-(4-Chloro-phenyl)-3R-[2,2-dimethyl-1S-(5-phenyl-[1,2,4]oxadiazol-3-yl)-propylcarbamoyl]-hexanoicAcid

¹H-NMR; delta(CH3OD), 8.07 (2H, m), 7.61 (3H, m), 6.93 (4H, m), 5.15(1H, s), 2.94 (1H, m), 2.5 (4H, m), 1.5 (4H, m) and 1.07 (9H, s).

¹³C-NMR; delta (CH3OD), 178.0, 177.1, 142.6, 134.6, 132.7, 131.0, 130.8,129.5, 129.4, 125.7, 55.7, 43.8, 39.0, 36.3, 36.1, 34.1, 30.3 and 27.4.

LRMS; +ve ion 506.2 (M+Na), −ve ion 482.4 (M−H).

Also prepared, the diastereomer6-(4-Chloro-phenyl)-3R-[2,2-dimethyl-1R-(5-phenyl-[1,2,4]oxadiazol-3-yl)-propylcarbamoyl]-hexanoicAcid

M+H=485, M+Na=507.2, M−H=482.6.

The compounds of Examples 29 and 30 were prepared by the method ofExample 1.

Example 293R-[2,2-Dimethyl-1S-(5-thiophen-2-yl-[1,2,4]oxadiazol-3-yl)-propylcarbamoyl]-2S-hydroxy-5-methyl-hexanoicAcid

¹H-NMR; delta (CH3OD), 7.95 (1H, m), 7.87 (1H, d, J=5.0 Hz), 7.28 (1H,m), 5.15 (1H, s), 4.18 (2H, d, J=6.4 Hz), 2.94 (1H, m), 1.68 (1H, m),1.48 (1H, m), 1.31 (1H, m), 1.06 (9H, s), 0.88 (3H, d, J=6.4 Hz) and0.82 (3H, d, J=6.5 Hz).

LRMS; −ve ion 408.2 (M−H).

Example 303R-[1S-(5-Furan-2-yl-[1,2,4]oxadiazol-3-yl)-2,2-dimethyl-propylcarbamoyl]-2S-hydroxy-5-methyl-hexanoicAcid

¹H-NMR; delta (CH3OD), 7.88 (1H, s), 7.45 (1H, d, J=3.6 Hz), 6.74 (1H,m), 5.15 (1H, s), 4.18 (2H, d, J=6.4 Hz), 2.91 (1H, m), 1.65 (1H, m),1.50 (1H, m), 1.31 (1H, m), 1.06 (9H, s), 0.88 (3H, d, J=6.4 Hz) and0.82 (3H, d, J=6.5 Hz).

LRMS; −ve ion 392.2 (M−H).

The compounds of Example 31 and 32 were prepared by the method ofExample 2. The synthesis to the appropriate chiral succinate in Step Eis detailed within WO 94/02446 using the appropriate cinnamyl bromide orcyclopentylmethyl iodide instead of the methallyl iodide as detailed inthe aforementioned patent.

Example 31N₄-[2,2-Dimethyl-1S-(3-phenyl-[1,2,4]oxadiazol-5-yl)-propyl]-2S,N₁-dihydroxy-3R-(3-phenyl-allyl)-succinamide

¹H-NMR; delta(CH3OD), 7.95 (2H, d, J=7.2 Hz), 7.53 (1H, m), 7.48 (2H,m), 7.09 (2H, d, J=6.4 Hz), 6.91 (3H, m), 6.31 (1H, d, J=15.8 Hz), 6.04(1H, m), 5.26 (1H, s), 4.14 (1H, d, J=7.6 Hz), 3.02 (1H, m), 2.46 (1H,m), 2.37 (1H, m) and 1.07 (9H, s).

¹³C-NMR; delta (CH3OD), 179.8, 175.9, 172.0, 169.6, 138.8, 134.0, 132.8,130.4, 129.7, 128.9, 128.4, 128.4, 127.3, 73.2, 56.5, 51.3, 36.8 and34.0.

LRMS; +ve ion 501.2 (M+Na), −ve ion 477.4 (M−H).

Example 322R-Cyclopentylmethyl-3S,N₄-dihydroxy-N₁-[1S-(3-isopropyl-[1,2,4]oxadiazol-5-yl)-2,2-dimethyl-propyl]-succinamide

¹H-NMR; delta(CH₃₀D), 5.13 (1H, s), 3.99 (1H, d, J=7.7 Hz), 3.06 (1H,m), 2.87 (1H, m), 1.83 (1H, m), 1.72 (1H, m), 1.63-1.39 (6H, bm), 1.31(6H, d, J=6.9 Hz), 1.27 (1H, m), 1.03 (9H, s) and 1.02 (2H, m). 13C-NMR; delta (CH3OD), 179.6, 176.6, 176.5, 172.0, 73.6, 56.8, 50.8,39.6, 36.7, 36.5, 34.7, 33.6, 28.3, 27.2, 26.5 and 21.2.

LRMS; +ve ion 411.2 (M+H), −ve ion 409.6 (M−H).

The compounds of Examples 33-35 were prepared by the method of Example 3using the appropriate aryl bromide in Step B.

Example 332R-[3-(3,5-Bis-trifluoromethyl-phenyl)-propyl]-N₁-[2,2-dimethyl-1S-(5-thiophen-2-yl-[1,2,4]oxadiazol-3-yl)-propyl]-3S,N₄-dihydroxy-succinamide

¹H-NMR; delta(CH3OD), 8.38 (1H, d, J=9.4 Hz), 7.86 (1H, s), 7.75 (3H,bs), 7.4 (1H, d, J=3.5 Hz), 6.7 (1H, m), 5.12 (1H, d, J=9.4 Hz), 4.26(1H, d, J=4.0 Hz), 2.8 (3H, bm), 1.8 (4H, bm) and 1.0 (9H, s).

LRMS; +ve ion 623.2 (M+H), −ve ion 621.0 (M−H).

Example 342R-[3-(3,5-Bis-trifluoromethyl-phenyl)-propyl]-N₁-[1S-(5-furan-2-yl-[1,2,4]oxadiazol-3-yl)-2,2-dimethyl-propyl]-3S,N₄-dihydroxy-succinamide

¹H-NMR; delta(CH3OD), 8.38 (1H, d, J=9.4 Hz), 7.86 (1H, s), 7.75 (3H,bs), 7.4 (1H, d, J=3.5 Hz), 6.7 (1H, m), 5.12 (1H, d, J=9.4 Hz), 4.26(1H, d, J=4.0 Hz), 2.8 (3H, bm), 1.8 (4H, bm) and 1.0 (9H, s).

LRMS; +ve ion 629.4 (M+Na), −ve ion 605.4 (M−H).

Example 352R-[3-(4-Ethoxy-phenyl)-propyl]-N₁-[1S-(5-furan-2-yl-[1,2,4]oxadiazol-3-yl)-2,2-dimethyl-propyl]-3S,N₄-dihydroxy-succinamide

¹H-NMR; delta(CH3OD), 7.86 (2H, m), 7.25 (1H, dd, J=3.8 Hz), 6.83 (2H,d, J=8.6 Hz), 6.54 (2H, d, J=8.6 Hz), 5.14 (1H, s), 4.03 (1H, d, J=7.6Hz), 3.87 (2H, q, J=6.96, 14.0 Hz), 2.88 (1H, m), 2.45 (2H, bm), 1.53(4H, bm), 1.33 (3H, t, J=7.0 Hz) and 1.06 (9H, s).

LRMS; +ve ion 515.2 (M+H), −ve ion 513.2 (M−H).

The compound of Examples 36 was prepared by the method of Example 2. Thesynthesis to the appropriate chiral succinate in Step E is detailedwithin WO 01/10834.

Example 363-Cyclopentyl-N-[2,2-dimethyl-1S-(3-phenyl-[1,2,4]oxadiazol-5-yl)-propyl]-2R-[(formyl-hydroxy-amino)-methyl]-propionamide

¹H-NMR; delta(CH3OD), 8.26 (03H, s), 8.05 (2H, d, J=6.9 Hz), 7.84 (0.7H,s), 7.52 (3H, m), 5.20 (1H, m), 3.75 (1H, m), 3.63 (0.3H, dd, J=13.9,5.5 Hz), 3.43 (0.7H, dd, J=14.2, 4.6 Hz), 3.18 (0.7H, m), 3.00 (0.3H,m), 1.92 (1H, m), 1.47 (8H, m), 1.10 (3H, s), 1.08 (6H, s) and 0.98 (2H,m).

¹³C-NMR; delta (CH3D), 179.9, 176.9, 176.6, 169.3, 163.8, 159.2, 132.5,130.0, 129.6, 128.9, 128.3, 127.9, 56.8, 56.7, 53.9, 50.3, 44.8, 44.6,39.1, 38.9, 37.9, 37.7, 35.9, 35.8, 34.1, 33.4, 33.3, 26.9, 26.1 and25.9.

LRMS; +ve ion 451 (M+Na), −ve ion 427 (M−H).

The compound of Example 37 was prepared by the method of Example 1. Thesynthesis to the appropriate chiral succinate in Step E is detailedwithin WO 01/10834.

Example 373-Cyclopentyl-N-[2,2-dimethyl-1S-(5-phenyl-[1,2,4]oxadiazol-3-yl)-propyl]-2R-[(formyl-hydroxy-amino)-methyl]-propionamide

¹H-NMR; delta(CH3OD), 8.49 (0.6H, d, J=8.7 Hz), 8.37 (0.4H, d, J=8.1Hz), 8.28 (0.4H, s), 8.14 (2H, m), 7.85 (0.6H, s), 7.65 (1H, m), 7.59(2H, m), 4.81 (1H, s), 3.79 (1H, m), 3.63 (0.4H, m), 3.43 (0.6H, m),3.13 (0.6H, m), 2.97 (0.4H, m), 1.55 (9H, m), 1.08 (3H, s), 1.07 (6H, s)and 1.04 (2H, m).

¹³C-NMR; delta (CH3OD), 176.6, 171.6, 164.2, 159.7, 134.6, 132.8, 130.8,130.3, 129.4, 125.7, 69.5, 56.0, 54.3, 50.8, 45.4, 45.3, 40.6, 39.5,38.3, 38.2, 35.9, 34.5, 33.8, 33.7, 32.0, 27.5, 26.4 and 26.3.

LRMS; +ve ion 429 (M+H).

Biological Results A. Enzyme Inhibition Assays

Compounds of the invention were tested to assess their activities asinhibitors of MMP9 and MMP12.

MMP9 Assay Protocol

Compounds were tested for inhibitory activity against 92 kDa gelatinase(MMP9) in an assay using a coumarin-labelled peptide substrate,(7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L-2,3-diaminopropionyl)-Ala-Arg-NH₂(McaPLGLDpaAR) (Knight et al, FEBS Lett. 1992; 263-266).

Stock solutions were made up as follows:

-   -   Assay Buffer: 100 mM Tris-HCl pH 7.6 containing 100 mM NaCl, 10        mM CaCl₂, and 0.05% Brij 35    -   Substrate: 0.4 mM McaPLGLDpaAR (from Bachem) (0.437 mg/ml) stock        solution in 100% DMSO (stored at −20° C.). Dilute to 8 μM in        assay buffer.    -   Enzyme: Recombinant human 92 kDa gelatinase (MMP-9; APMA        (4-aminophenyl    -   mercuric acetate)—activated if necessary) appropriately diluted        in assay buffer.

Test Compounds were prepared initially as 10 mM compound solution in100% DMSO, diluted to 1 mM in 100% DMSO, then serially diluted 3-fold in100% DMSO across columns 1-10 of a 96-well microtitre plate Assayconcentration range, 100 μM (column 1) to 5.1 nM (column 10)

The assay was performed in a total volume of 100 μl per well in 96-wellmicrotitre plates. Activated enzyme (20 μl) was added to the wellsfollowed by 20 μl of assay buffer. Appropriate concentrations of testcompounds dissolved in 10 μl of DMSO were then added followed by 50 μlof McaPLGLDpaAR (8 μM, prepared by dilution of DMSO stock in assaybuffer). For each assay ten concentrations of test compound wereexamined in duplicate. Control wells lack either enzyme or testcompound. The reactions were incubated at 37° C. for 2 hours. Thefluorescence at 405 nm was measured immediately with an SLT Fluostarfluorometer (SLT Labinstruments GmbH, Grödig, Austria) using 320 nmexcitation, without stopping the reaction.

The effect of the test compound was determined from the dose responsecurve generated by the 10 duplicate concentrations of inhibitor. TheIC₅₀ (the concentration of compound required to give a 50% decrease inenzyme activity) was obtained by fitting data to the equation,Y=a+((b−a)/(1+(c/X)^(d))). (Y=inhibition achieved for a particular dose;X=the dose in nM; a=minimum y or zero % inhibition; b=maximum y or 100%inhibition; c=is the IC₅₀; d=is the slope). The result was rounded toone significant figure.

MMP12 Assay Protocol

Compounds were tested for inhibitory activity against metalloelastase(MMP12) in an assay using a coumarin-labelled peptide substrate,(7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L-2,3-diaminopropionyl)-Ala-Arg-NH₂(McaPLGLDpaAR) (Knight et al, FEBS Lett. 1992; 263-266). The protocolfor this assay was as described for the MMP9 assay above.

MMP1 Assay Protocol

-   -   Compounds were tested for inhibitory activity against        collagenase (MMP1) in an assay using a coumarin-labelled peptide        substrate,        (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L-2,3-diaminopropionyl)-Ala-Arg-NH₂        (McaPLGLDpaAR) (Knight et al, FEBS Lett. 1992; 263-266). The        protocol for this assay was as described for the MMP9 assay        above.

Results:

Example MMP9 MMP12 MMP1 Number IC50 (nM) IC50 (nM) IC50 (nM)  1 B A B  2B A B  3 A A D  4 B A B  5 B A B  6 C A B  7 C B C  8 B A B  9 C A B 10C A C 11 C A C 12 B A B 13 B A B 14 C A C 15 B A B 16 B A B 17 C A B 18B A B 19 B A B 20 A A B 21 A A B 22 Not tested Not tested A 23 A A B 24C A C 25 B A B 26 D D Not tested 27 D D Not tested 28 Not tested D Nottested 29 C B C 30 D C D 31 D B D 32 A A A 33 D B D 34 D D D 35 A A DKey to biological data Range A <100 nM B 100-1000 nM C 1000-10,000 nMD >10,000 nM

These results show that in general, the compounds tested were active asinhibitors of MMP12, with certain examples showing selective inhibitionof both MMP-9 and 12 relative to MMP-1.

B. CCl₄-Induced Liver Fibrosis Model

Carbon tetrachloride (CCl₄) induces liver fibrosis when administeredintraperitoneally (Bulbena O, Culat J, Bravo M L., Inflammation 1997October; 21(5):475-88). Compounds of the invention can be evaluated fortheir ability to prevent the CCl₄-induced formation of fibrotic tissue.

Animals

Male Sprague-Dawley rats, 7 weeks old, weight approx. 300 g from CharlesRiver/Iffa-Crédo, St-Germain/l'Arbresle, France.

Rats were acclimatised for 5 days before commencing experiments, inair-conditioned rooms, 2 animals per cage, Temperature: 22° C.±2,Relative humidity: 55%±10 Light: 12 hour cycle (7 a.m.-7 p.m.), Cage:Makrolone cage 42.5×26.6×15 on each fitted with a stainless steelcover-feed rack.

The study involved the following groups of 8 animals each, as indicatedbelow.

Group 1: “Sham” animals received CCl₄ vehicle (i.p.) and once daily, thevehicle of test substance (s.c.)Group 2: Positive control group received CCl₄ (i.p.), and once daily,the vehicle of the test substance (s.c.)Group 3: Experimental group received CCl₄ (i.p.), and once daily, 2mg/kg s.c. of the compound of Example 13.Group 4: Experimental group received CCl₄ (i.p.), and once daily, 10mg/kg s.c. of the compound of Example 13.Group 5: Experimental group received CCl₄ (i.p.) and once daily, 20mg/kg s.c. of the compound of Example 13.

Rats were labelled on their tails. The labels were checked and renewed,if necessary, after every CCl₄ injection.

Procedure

CCl₄ (Prolabo) in olive oil was administered every 3 days for threeweeks by intraperitoneal injection (0,25 ml CCl₄/kg body weight, dilutedin oil 1:1 vol:vol for a total volume of 0.5 ml/kg). Animals wereweighed daily. If body weight decreased by more than 10% of the initialweight, the animal was excluded from the study.

Vehicles and compound were used as follows:

-   -   CCl₄ was administered in olive oil (prolabo) at a 1:1 dilution;    -   The compound of Example 13 was suspended in 0.25% Tween-80 and        0.25% carboxymethylcellulose in sterile 0.9% NaCl. The solution        was kept at 4° C. throughout the experiment and used each day to        prepare the suspensions.

The compound of Example 13 was administered daily by subcutaneous (s.c.)injection at a volume of administration of 5 ml/kg. Groups 1 and 2 weredosed s.c. with 5 ml/kg of vehicle. Freshly prepared solutions were usedon each day of the experiment. Administrations were carried out each dayat the same time.

The treatment of groups of this study was started for each animal at thetime of the first CCl₄ administration and was continued for 21consecutive days. The last administration of test substances or vehiclewas done 1 day before the sacrifice of the animals.

Results

Death was reported for 16 animals. Date and supposed cause are reportedin Table 1.

Serum Enzyme Levels

Animals were killed 21 days following the first CCl₄ administration byisofurane inhalation. Blood was withdrawn individually at the time ofsacrifice, i.e. one day after the last administration of test substanceor vehicle. Blood was centrifuged at 4° C. Plasma was carefullycollected and aliquoted in 3 fractions. Plasma aspartate aminotransferase (ASAT) and alanine amino transferase (ALAT) levels weremeasured in order to assess liver necrosis. Increased ASAT and ALATlevels in serum are associated with liver impairment. Average ASAT andALAT levels for control animals and those treated with the compound ofExample 13 at three different dosages are shown in FIG. 1 (Y-axis isunits of enzyme activity per litre blood, IU/L). Subcutaneous treatmentwith the compound of Example 13 clearly decreases ASAT and ALAT levelscompared to animals treated with vehicle. This demonstrates that thecompound of Example 13 has a protective effect on the liver.

Histological Evaluation of Liver Fibrosis

Liver fibrosis was evaluated by measuring the area of fibrosis in theliver using microchotomy. Results are reported as percentage area thatwas fibrotic.

The liver was removed, the three lobes were dissected and samples wereremoved and either fixed in 10% formaldehyde or frozen at −80° C.

Liver sections were embedded in paraffin blocks. Sectioning and stainingwith Sirius red was performed. Quantification of the fibrosis in liverwas carried out on a minimum of 3 sections taken from differentlocations in the liver. The quantitative analysis was performed using animage analyser (Imstar) and the software Morphostar.

Average area percentages of fibrosis in the livers of animals in thedifferent groups were calculated, and the results are shown in FIG. 2.

B. IL2-Induced Peritoneal Recruitment of Lymphocytes

Administration of IL2 intraperitoneally causes migration of lymphocytesinto the intraperitoneal cavity. This is a model for the cellularmigration that occurs during inflammation.

Compounds of the invention inhibit IL2-induced lymphocyte recruitment.

Protocol

C3H/HEN mice (Elevage Janvier, France) were intraperitoneally injectedwith IL2 (Serono Pharmaceutical Research Institute, 20 μg/kg, insaline). Compounds of the invention were suspended in 0.5%carboxymethylcellulose (CMC)/0.25% tween-20 and were administered by scor po route (10 ml/kg) 15 min prior to administration of IL2.

Twenty-four hours after administration of IL2, peritoneal white bloodcells were collected by 3 successive lavages of the peritoneal cavitywith 5 ml phosphate buffered saline (PBS)-1 mM EDTA (+4° C.). Thesuspension was centrifuged (1700 g×10 min at +4° C.). The resultingpellet was suspended in 1 ml PBS-1 mM EDTA.

Lymphocytes were identified and counted using a Beckman/Coulter counter.

Experimental Design

The animals were divided into 5 groups (6 mice each group):

Group 1: (baseline) received 0.5% CMC/0.25% tween-20 (vehicle ofcompound of the invention) and saline (vehicle of IL2);Group 2: (control IL2) received 0.5% CMC/0.25% tween-20 and injection ofIL2;Group 3: Experimental group (Compound of the invention Dose 1) receiveda compound of the invention and injection of IL2;Group 4: Experimental group (Compound of the invention Dose 2) receiveda compound of the invention and injection of IL2;Group 5: Experimental group (Compound of the invention Dose 3) receiveda compound of the invention and injection of IL2;Group 6: Reference group received reference compound dexamethasone andinjection of IL2

Calculation

Inhibition of lymphocyte recruitment was calculated as follows:

${\% \mspace{14mu} {inhibition}} = {\frac{1 - \left( {{LyX} - {{Ly}\; 1}} \right)}{\left( {{{Ly}\; 2} - {{Ly}\; 1}} \right)} \times 100\%}$

Where Ly 1=Number of lymphocytes in group 1 (E3/μl), Ly 2=Number oflymphocytes in group 2 (E3/μl), Ly X=Number of lymphocytes in group X(3-5) (E3/μl)

The dose of compound of the invention required to inhibit lymphocyterecruitment by 50% (ID50) was calculated using a curve-fitting routine.Results are listed in Table 1.

TABLE 1 ID₅₀ for inhibition of IL2-induced peritoneal recruitment oflymphocytes by compounds of the invention Dose range or ID₅₀ Exampledoses (mg/kg) Route (mg/kg) dexamethasone 0.1-1 Subcutaneous 0.05Example 13 0.03, 0.3, 3, 30 Subcutaneous 0.1 Example 13 0.3, 3, 30 Oral1 Example 5 0.3, 1, 3, 10, Subcutaneous 1 30

1. A method of treatment or prophylaxis of diseases mediated by MMPs inmammals comprising administering to the mammal an effective amount of acompound having formula (IA) or (IB)

wherein w represents HO(C═O)—, HONH(C═O)— or H(C═O)N(OH)—; X represents—O— or —S—; R₁ represents hydrogen; —OH or —SH; fluoro or chloro; —CF₃;(C₁-C₆)alkyl; (C₁-C₆)alkoxy; (C₂-C₆)alkenyl; phenyl or substitutedphenyl; phenyl (C₁-C₆)alkyl or substituted phenyl(C₁-C₆)alkyl; phenyl(C₂-C₆)alkenyl or substituted phenyl(C₂-C₆)alkenyl heterocyclyl orsubstituted heterocyclyl; heterocyclyl(C₁-C₆)alkyl or substitutedheterocyclyl(C₁-C₆)alkyl; a group BSO_(n)A- wherein n is 0, 1 or 2 and Bis hydrogen or a (C₁-C₆) alkyl, phenyl, substituted phenyl,heterocyclyl, substituted heterocyclyl, (C₁-C₆)acyl, phenacyl orsubstituted phenacyl group, and A represents (C₁-C₆)alkylene; —NH₂,(C₁-C₆)alkylamino or di(C₁-C₆)alkylamino; amino(C₁-C₆)alkyl,(C₁-C₆)alkylamino(C₁-C₆)alkyl, di(C₁-C₆)alkylamino(C₁-C₆)alkyl,hydroxy(C₁-C₆)alkyl, mercapto(C₁-C₆)alkyl or carboxy(C₁-C₆) alkylwherein the amino-, hydroxy-, mercapto- or carboxyl-group are optionallyprotected or the carboxyl-group amidated; or a cycloalkyl, cycloalkenylor non-aromatic heterocyclic ring containing up to 3 heteroatoms, any ofwhich may be (i) substituted by one or more substituents selected fromC₁-C₆ alkyl, C₂-C₆ alkenyl, halo, cyano (—CN), —CO₂H, —CO₂R, —CONH₂,—CONHR, —CON(R)₂, —OH, —OR, oxo-, —SH, —SR, —NHCOR, and —NHCO₂R whereinR is C₁-C₆ alkyl or benzyl and/or (ii) fused to a cycloalkyl orheterocyclic ring; R₂ represents a group R₁₀—(X₁)_(p)-(ALK)_(m)— whereinR₁₀ represents hydrogen, or a C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,cycloalkyl, aryl, or heterocyclyl group, any of which may beunsubstituted or substituted by (C₁-C₁₂)alkyl, (C₁-C₁₂)alkoxy, hydroxy,mercapto, (C₁-C₁₂)alkylthio, amino, halo (including fluoro, chloro,bromo and iodo), trifluoromethyl, cyano, nitro, oxo, —COOH, —CONH₂,COOR^(A), —NHCOR^(A), CONHR^(A), —NHR^(A), —NR^(A)R^(B), or—CONR^(A)R^(B) wherein R^(A) and R^(B) are independently a (C₁-C₁₂)alkylgroup and ALK represents a straight or branched divalent C₁-C₆ alkylene,C₂-C₆ alkenylene, or C₂-C₆ alkynylene radical, and may be interrupted byone or more non-adjacent —NH—, —O— or —S-linkages, X₁ represents —NH—,—O— or —S—, —NR^(c) or —NCOR^(c) wherein R^(c) is a (C₁-C₁₂)alkyl group,and m and p are independently 0 or 1; R₃ is C₁-C₆ alkyl, phenyl, 2,3-,or 4-pyridyl, 2- or 3-thienyl, 2,-3-, or 4-hydroxyphenyl, 2,-3-, or4-methoxyphenyl, 2,3-, or 4-pyridylmethyl, benzyl, 2,3-, or4-hydroxybenzyl, 2,-3-, or 4-benzyloxybenzyl, 2,-3-, or 4-C₁-C₆alkoxybenzyl, or benzyloxy(C₁-C₆ alkyl)-; or the characterizing group ofa natural α-amino acid, in which any functional group may be protected,any amino group may be acylated and any carboxyl group present may beamidated; or a group -[Alk]_(n)R₆ where Alk is a (C₁-C₆)alkyl or(C₂-C₆)alkenyl group optionally interrupted by one or more —O—, or —S—atoms or —N(R₇) groups [where R₇ is a hydrogen atom or a (C₁-C₆)alkylgroup], n is 0 or 1, and R₆ is an optionally substituted cycloalkyl orcycloalkenyl group; or a benzyl group substituted in the phenyl ring bya group of formula —OCH₂COR₈ where R₈ is hydroxyl, amino, (C₁-C₆)alkoxy,phenyl(C₁-C₆)alkoxy, (C₁-C₆)alkylamino, di((C₁-C₆)alkyl)amino,phenyl(C₁-C₆)alkylamino, the residue of an amino acid or acid halide,ester or amide derivative thereof, said residue being linked via anamide bond, said amino acid being selected from glycine, α or β alanine,valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan,serine, threonine, cysteine, methionine, asparagine, glutamine, lysine,histidine, arginine, glutamic acid, and aspartic acid; or aheterocyclic(C₁-C₆)alkyl group, either being unsubstituted or mono- ordi-substituted in the heterocyclic ring with halo, nitro, carboxy,(C₁-C₆)alkoxy, cyano, (C₁-C₆)alkanoyl, trifluoromethyl (C₁-C₆)alkyl,hydroxy, formyl, amino, (C₁-C₆)alkylamino, di-(C₁-C₆)alkylamino,mercapto, (C₁-C₆)alkylthio, hydroxy(C₁-C₆)alkyl, mercapto(C₁-C₆)alkyl or(C₁-C₆)alkylphenylmethyl; or a group —CR_(a)R_(b)R_(c) in which: each ofR_(a), R_(b) and R_(c) is independently hydrogen, (C₁-C₆)alkyl,(C₂-C₆)alkenyl, (C₂-C₆)alkynyl, phenyl(C₁-C₆)alkyl, (C₃-C₈)cycloalkyl;or R_(c) is hydrogen and R_(a) and R_(b) are independently phenyl orheteroaryl such as pyridyl; or R_(c) is hydrogen, (C₁-C₆)alkyl,(C₂-C₆)alkenyl, (C₂-C₆)alkynyl, phenyl(C₁-C₆)alkyl, or(C₃-C₈)cycloalkyl, and R_(a) and R_(b) together with the carbon atom towhich they are attached form a 3 to 8 membered cycloalkyl or a 5- to6-membered heterocyclic ring; or R_(a), R_(b) and R_(c) together withthe carbon atom to which they are attached form a tricyclic ring (forexample adamantyl); or R_(a) and R_(b) are each independently(C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, phenyl(C₁-C₆)alkyl, or agroup as defined for R_(b) below other than hydrogen, or R_(a) and R_(c)together with the carbon atom to which they are attached form acycloalkyl or heterocyclic ring, and R_(b) is hydrogen, —OH, —SH,halogen, —CN, —CO₂H, (C₁-C₄)perfluoroalkyl, —CH₂OH, —CO₂(C₁-C₆)alkyl,—O(C₁-C₆)alkyl, —O(C₂-C₆)alkenyl, —S(C₁-C₆)alkyl, —SO(C₁-C₆)alkyl,—SO₂(C₁-C₆) alkyl, —S(C₂-C₆)alkenyl, —SO(C₂-C₆)alkenyl,—SO₂(C₂-C₆)alkenyl or a group -Q-W wherein Q represents a bond or —O—,—S—, —SO— or —SO₂— and W represents a phenyl, phenylalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkylalkyl, (C₄-C₈)cycloalkenyl,(C₄-C₈)cycloalkenylalkyl, heteroaryl or heteroarylalkyl group, whichgroup W may optionally be substituted by one or more substituentsindependently selected from, hydroxyl, halogen, —CN, —CO₂H,—CO₂(C₁-C₆)alkyl, —CONH₂, —CONH(C₁-C₆)alkyl, —CONH(C₁-C₆ alkyl)₂, —CHO,—CH₂OH, (C₁-C₄)perfluoroalkyl, —O(C₁-C₆)alkyl, —S(C₁-C₆)alkyl,—SO(C₁-C₆)alkyl, —SO₂(C₁-C₆)alkyl, —NO₂, —NH₂, —NH(C₁-C₆)alkyl,—N((C₁-C₆)alkyl)₂, —NHCO(C₁-C₆)alkyl, (C₁-C₆)alkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, (C₃-C₆)cycloalkyl, (C₄-C₈)cycloalkenyl, phenyl orbenzyl; R₄ represents optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₁-C₃ perfluoroalkyl, cycloalkyl, cycloalkyl(C₁-C₆alkyl), cycloalkenyl, cycloalkenyl(C₁-C₆ alkyl)-, phenyl, phenyl(C₁-C₆alkyl)-, naphthyl, non-aryl heterocyclyl, non-aryl heterocyclyl(C₁-C₆)alkyl-, heteroaryl; or heteroaryl(C₁-C₆ alkyl)-; or apharmaceutically acceptable salt, hydrate or solvate thereof.
 2. Amethod for the preparation of a medicament for the treatment orprophylaxis of diseases mediated by MMPs comprising adding to amedicament formulation a compound having formula (IA) or (IB)

wherein W represents HO(C═O)—, HONH(C═O)— or H(C═O)N(OH)—; X represents—O— or —S—; R₁, represents hydrogen; —OH or —SH; fluoro or chloro; —CF₃;(C₁-C₆)alkyl; (C₁-C₆)alkoxy; (C₂-C₆)alkenyl; phenyl or substitutedphenyl; phenyl (C₁-C₆)alkyl or substituted phenyl(C₁-C₆)alkyl; phenyl(C₂-C₆)alkenyl or substituted phenyl(C₂-C₆)alkenyl heterocyclyl orsubstituted heterocyclyl; heterocyclyl(C₁-C₆)alkyl or substitutedheterocyclyl(C₁-C₆)alkyl; a group BSO_(n)A- wherein n is 0, 1 or 2 and Bis hydrogen or a (C₁-C₆) alkyl, phenyl, substituted phenyl,heterocyclyl, substituted heterocyclyl, (C₁-C₆)acyl, phenacyl orsubstituted phenacyl group, and A represents (C₁-C₆)alkylene; —NH₂,(C₁-C₆)alkylamino or di(C₁-C₆)alkylamino; amino(C₁-C₆)alkyl,(C₁-C₆)alkylamino(C₁-C₆)alkyl, di(C₁-C₆)alkylamino(C₁-C₆)alkyl,hydroxy(C₁-C₆)alkyl, mercapto(C₁-C₆)alkyl or carboxy(C₁-C₆) alkylwherein the amino-, hydroxy-, mercapto- or carboxyl-group are optionallyprotected or the carboxyl-group amidated; or a cycloalkyl, cycloalkenylor non-aromatic heterocyclic ring containing up to 3 heteroatoms, any ofwhich may be (i) substituted by one or more substituents selected fromC₁-C₆ alkyl, C₂-C₆ alkenyl, halo, cyano (—CN), —CO₂H, —CO₂R, —CONH₂,—CONHR, —CON(R)₂, —OH, —OR, oxo-, —SH, —SR, —NHCOR, and —NHCO₂R whereinR is C₁-C₆ alkyl or benzyl and/or (ii) fused to a cycloalkyl orheterocyclic ring; R₂ represents a group R₁₀—(X₁)_(p)-(ALK)_(m)— whereinR₁₀ represents hydrogen, or a C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,cycloalkyl, aryl, or heterocyclyl group, any of which may beunsubstituted or substituted by (C₁-C₁₂)alkyl, (C₁-C₁₂)alkoxy, hydroxy,mercapto, (C₁-C₁₂)alkylthio, amino, halo (including fluoro, chloro,bromo and iodo), trifluoromethyl, cyano, nitro, oxo, —COOH, —CONH₂,COOR^(A), —NHCOR^(A), —CONHR^(A), —NHR^(A), —NR^(A)R^(B), or—CONR^(A)R^(B) wherein R^(A) and R^(B) are independently a (C₁-C₁₂)alkylgroup and ALK represents a straight or branched divalent C₁-C₆ alkylene,C₂-C₆ alkenylene, or C₂-C₆ alkynylene radical, and may be interrupted byone or more non-adjacent —NH—, —O— or —S-linkages, X₁ represents —NH—,—O— or —S—, —NR^(c) or —NCOR^(c) wherein R^(c) is a (C₁-C₁₂)alkyl group,and m and p are independently 0 or 1; R₃ is C₁-C₆ alkyl, phenyl, 2,3-,or 4-pyridyl, 2- or 3-thienyl, 2,-3-, or 4-hydroxyphenyl, 2,-3-, or4-methoxyphenyl, 2,3-, or 4-pyridylmethyl, benzyl, 2,3-, or4-hydroxybenzyl, 2,-3-, or 4-benzyloxybenzyl, 2,-3-, or 4-C₁-C₆alkoxybenzyl, or benzyloxy(C₁-C₆ alkyl)-; or the characterizing group ofa natural α-amino acid, in which any functional group may be protected,any amino group may be acylated and any carboxyl group present may beamidated; or a group -[Alk]_(n)R₆ where Alk is a (C₁-C₆)alkyl or(C₂-C₆)alkenyl group optionally interrupted by one or more —O—, or —S—atoms or —N(R₇) groups [where R₇ is a hydrogen atom or a (C₁-C₆)alkylgroup], n is 0 or 1, and R₆ is an optionally substituted cycloalkyl orcycloalkenyl group; or a benzyl group substituted in the phenyl ring bya group of formula —OCH₂COR₅ where R₈ is hydroxyl, amino, (C₁-C₆)alkoxy,phenyl(C₁-C₆)alkoxy, (C₁-C₆)alkylamino, di((C₁-C₆)alkyl)amino,phenyl(C₁-C₆)alkylamino, the residue of an amino acid or acid halide,ester or amide derivative thereof, said residue being linked via anamide bond, said amino acid being selected from glycine, α or β alanine,valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan,serine, threonine, cysteine, methionine, asparagine, glutamine, lysine,histidine, arginine, glutamic acid, and aspartic acid; or aheterocyclic(C₁-C₆)alkyl group, either being unsubstituted or mono- ordi-substituted in the heterocyclic ring with halo, nitro, carboxy,(C₁-C₆)alkoxy, cyano, (C₁-C₆)alkanoyl, trifluoromethyl (C₁-C₆)alkyl,hydroxy, formyl, amino, (C₁-C₆)alkylamino, di-(C₁-C₆)alkylamino,mercapto, (C₁-C₆)alkylthio, hydroxy(C₁-C₆)alkyl, mercapto(C₁-C₆)alkyl or(C₁-C₆)alkylphenylmethyl; or a group —CR_(a)R_(b)R_(c) in which: each ofR_(a), R_(b) and R_(c) is independently hydrogen, (C₁-C₆)alkyl,(C₂-C₆)alkenyl, (C₂-C₆)alkynyl, phenyl(C₁-C₆)alkyl, (C₃-C₈)cycloalkyl;or R_(c) is hydrogen and R_(a) and R_(b) are independently phenyl orheteroaryl such as pyridyl; or R_(c) is hydrogen, (C₁-C₆)alkyl,(C₂-C₆)alkenyl, (C₂-C₆)alkynyl, phenyl(C₁-C₆)alkyl, or(C₃-C₈)cycloalkyl, and R_(a) and R_(b) together with the carbon atom towhich they are attached form a 3 to 8 membered cycloalkyl or a 5- to6-membered heterocyclic ring; or R_(a), R_(b) and R_(c) together withthe carbon atom to which they are attached form a tricyclic ring (forexample adamantyl); or R_(a) and R_(b) are each independently(C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, phenyl(C₁-C₆)alkyl, or agroup as defined for R_(c) below other than hydrogen, or R_(a) and R_(b)together with the carbon atom to which they are attached form acycloalkyl or heterocyclic ring, and R_(c) is hydrogen, —OH, —SH,halogen, —CN, —CO₂H, (C₁-C₄)perfluoroalkyl, —CH₂OH, —CO₂(C₁-C₆)alkyl,—O(C₁-C₆)alkyl, —O(C₂-C₆)alkenyl, —S(C₁-C₆)alkyl, —SO(C₁-C₆)alkyl,—SO₂(C₁-C₆) alkyl, —S(C₂-C₆)alkenyl, —SO(C₂-C₆)alkenyl,—SO₂(C₂-C₆)alkenyl or a group -Q-W wherein Q represents a bond or —O—,—S—, —SO— or —SO₂— and W represents a phenyl, phenylalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkylalkyl, (C₄-C₈)cycloalkenyl,(C₄-C₈)cycloalkenylalkyl, heteroaryl or heteroarylalkyl group, whichgroup W may optionally be substituted by one or more substituentsindependently selected from, hydroxyl, halogen, —CN, —CO₂H,—CO₂(C₁-C₆)alkyl, —CONH₂, —CONH(C₁-C₆)alkyl, —CONH(C₁-C₆ alkyl)₂, —CHO,—CH₂OH, (C₁-C₄)perfluoroalkyl, —O(C₁-C₆)alkyl, —S(C₁-C₆)alkyl,—SO(C₁-C₆)alkyl, —SO₂(C₁-C₆)alkyl, —NO₂, —NH₂, —NH(C₁-C₆)alkyl,—N((C₁-C₆)alkyl)₂, —NHCO(C₁-C₆)alkyl, (C₁-C₆)alkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, (C₃-C₆)cycloalkyl, (C₄-C₈)cycloalkenyl, phenyl orbenzyl; R₄ represents optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₁-C₃ perfluoroalkyl, cycloalkyl, cycloalkyl(C₁-C₆alkyl), cycloalkenyl, cycloalkenyl(C₁-C₆ alkyl)-, phenyl, phenyl(C₁-C₆alkyl)-, naphthyl, non-aryl heterocyclyl, non-aryl heterocyclyl(C₁-C₆)alkyl-, heteroaryl; or heteroaryl(C₁-C₆ alkyl)-; or apharmaceutically acceptable salt, hydrate or solvate thereof.
 3. Themethod according to claim 1 wherein the disease is selected from thegroup consisting of bone resorption, tumour growth or invasion bysecondary metastases, rheumatoid arthritis, septic arthritis,osteoarthritis, periodontitis, gingivitis, corneal ulceration,neuroinflammatory disorders, restenosis, emphysemia, fibrotic diseases,chronic obstructive pulmonary disease, bronchitis, asthma, autoimmunedisease, transplant rejection, cystic fibrosis, psoriasis, psodaticarthritis, degenerative cartilage loss, inflammatory gastric conditions,inflammatory bowel disease, and ulcerative colitis, atopic dermatitis,epidermolysis bullosa, epidermic ulceration, a neuropathy ornephropathy, lomerulonephriris and renal failure; ocular inflammation;liver cirrhosis, Sjoegren's syndrome; and an inflammatory condition ofthe nervous system.
 4. The method according to claim 1 wherein thedisease is selected from the group consisting of multiple sclerosis,emphysema, liver fibrosis, cystic fibrosis, chronic obstructivepulmonary disease, Crohn's disease, inflammatory bowel disease, andliver sclerosis.
 5. The method according to claim 1 wherein the diseaseis hepatitis.
 6. The method according to claim 2, wherein the disease isselected from the group consisting of bone resorption, tumour growth orinvasion by secondary metastases, rheumatoid arthritis, septicarthritis, osteoarthritis, periodontitis, gingivitis, cornealulceration, neuroinflammatory disorders, restenosis, emphysemia,fibrotic disease, chronic obstructive pulmonary disease, bronchitis,asthma, autoimmune disease, transplant rejection, cystic fibrosis,psoriasis, psodatic arthritis, degenerative cartilage loss, inflammatorygastric conditions, inflammatory bowel disease, and ulcerative colitis,atopic dermatitis, epidermolysis bullosa, epidermic ulceration, aneuropathy or nephropathy, glomerulonephritis and renal failure, ocularinflammation, liver cirrhosis, Sjoegren's syndrome, and an inflammatorycondition of the nervous system.
 7. The method according to claim 2,wherein the disease is selected from the group consisting of multiplesclerosis, emphysema, liver fibrosis, cystic fibrosis, chronicobstructive pulmonary disease, Crohn's disease, inflammatory boweldisease, and liver sclerosis.
 8. The method according to claim 2,wherein the disease is hepatitis.